title: FACS-sorted cKIT(+) spermatogonia, Cbx2[23KRA/23KRA], rep5

city: Ithaca

first: Jongmin

middle: J

last: Kim

state: NY

address: 930 Campus Road

department: Biomedical Sciences

institute: Cornell University

data_processing:
For Cbx2KO experiments, low quality reads (Phred score 20) and adapter containing reads (stringency 1) were trimmed using trim_galore (ver. 0.4.3) (Martin 2011). Trimmed reads were mapped to the mouse genome (mm10) with default parameters using STAR (ver. 2.5.3) (Dobin et al. 2013). Mapped reads were filtered with quality score 10 (-q 10), and only properly paired reads were retained (-f 2) using Samtools (ver. 1.4.1) (Li et al. 2009). Counts per gene was obtained using featureCounts function of the subread package (ver. 1.5.0) (Liao et al. 2014). Data from Cbx223KRA experiments were processed identically as the Cbx2KO experiment, except counts per gene were obtained with the strand parameter ‘-s 2’ of featureCounts to account for the stranded library.
Assembly: mm10
Supplementary files format and content: Tab-separated counts files

source_name: Testes

growth_protocol: Mice were maintained in shoebox cages with standard chow diet.

extract_protocol:
More than 100,000 c-KIT(+) cells were isolated, centrifuged to remove sorting buffer. Cell pellets were snap frozen in liquid nitrogen and stored in -80 ºC. RNA was isolated using Trizol-based phase separation, followed by column-based purification (Qiagen RNeasy mini, Cat# 74104).
We used SMARTer Stranded Total RNA-Seq Kit v2 (Takara, Cat# 634411) following manufacturer’s manual to generate RNA-seq libraries for Cbx2[23KRA] experiments. Cells from five control (Cbx2+/+) and five experimental (Cbx223KRA/23KRA) animals were used. cDNA was amplified with 5 PCR cycles. Subsequently, ribosomal cDNA was depleted, and unfragmented remaining cDNA was indexed and amplified with 16 PCR cycles.

treatment_protocol: Tamoxifen (Sigma-Aldrich, Cat# T5648) was dissolved in corn oil (Spectrum Chemical, Cat# CO136) to a final 20 mg/mL by incubating the mixture at 37 ºC overnight while rotating. Dissolved tamoxifen was filtered and aliquots for daily use were stored in sterile tubes and kept at 4 ºC for up to 4 days. 200 µL of warmed-up tamoxifen-corn oil was injected intraperitoneally daily for 3 consecutive days. For mock treatment, 200 µL of corn oil without Tamoxifen was injected.

value: Testes

value: cKIT(+) differentiating spermatogonia


title: FACS-sorted cKIT(+) spermatogonia, Cbx2[+/+], rep5

status: Public on Aug 14 2023

type: SRA

country: USA

library_source: transcriptomic

molecule: polyA RNA

organism: Mus musculus

extract_protocol:
extract protocol
We used SMARTer Stranded Total RNA-Seq Kit v2 (Takara, Cat# 634411) following manufacturer’s manual to generate RNA-seq libraries for Cbx2[23KRA] experiments. Cells from five control (Cbx2+/+) and five experimental (Cbx223KRA/23KRA) animals were used. cDNA was amplified with 5 PCR cycles. Subsequently, ribosomal cDNA was depleted, and unfragmented remaining cDNA was indexed and amplified with 16 PCR cycles.

tag: tissue

tag: cell type

tag: strain

tag: genotype

tag: treatment


title: T. gondii in vitro control conditions 2

city: Pittsburgh

first: Jon

middle: P

last: Boyle

state: PA

address: 4249 Fifth Avenue

department: Biological Sciences

institute: University of Pittsburgh

data_processing:
For H. hammondi vs. T. gondii comparisons, data were reduced to include only genes that could be matched between T. gondii and H. hammondi based on synteny.  "Species specific" genes are included in the counts file but not subject to further analysis
Assembly: T. gondii and H. hammondia verson 43
Supplementary files format and content: Counts files have raw counts for each transcripts
Supplementary files format and content: Analyzed files have statistical analyses and log-transformed data

organism: Toxoplasma gondii

source_name: freshly excysted sporozoites

growth_protocol: in vitro derived tachyzoites or in vitro derived sporozoites

extract_protocol:
Qiagen Rneasy
NEB Next or Illuina Tru Seq stranded mRNA
200 bp insert

treatment_protocol: control conditions or high pH, low C02, low serum conditions

value: freshly excysted sporozoites

value: sporozoites grown in vitro

value: VEG

value: control media conditions


title: FACS-sorted cKIT(+) spermatogonia, Cbx2[+/+], rep4


title: wt-kidney, rep 1, scRNA-seq

status: Public on Aug 15 2023

city: nanjing

first: XU

last: Ying

state: jiangsu

address: xuanwu district

country: China

institute: Nanjing university

description: 10x Genomics

library_source: transcriptomic single cell

data_processing:
scRNA-seq samples: the demultiplexing, barcoded processing, gene counting and aggregation were made using the Cell Ranger software v3.0.2 (https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/what-is-cell-ranger).
RNA-seq samples: CLC Genomics Workbench v 11.0.1, sequence reads were trimmed for adaptor sequence/low-quality sequence using CLC genomic benchwork (parameter- Quality limit: 0.05), trimmed sequence reads were mapped to  hg38 using CLC genomic benchwork (parameters- mismath cost: 2 insertion cost: 3 deletion cost: 3 length fraction: 0.8 similarity fraction: 0.8), read count extraction and normalization were performed using CLC genomic benchwork.
Assembly: mm10, hg38
Supplementary files format and content: Tab-separated values files and matrix files for scRNA-seq samples.
Supplementary files format and content: Tab-delimited text files include RPKM values for RNA-seq samples.

molecule: total RNA

source_name: kidney

growth_protocol: HMC and HRPC were  maintained in DMEM supplemented with 10% fetal bovine serum (FBS) in humidified atmosphere with 5% CO2 at 37℃.

extract_protocol:
21 weeks old wide type (wt) and leptin receptor-deficient (db/db) in C57BLKs/J background mice were sacrificed. Kidneys were perfused with PBS, followed by dynabead perfusion. Minced kidneys were digested with 1 mg/ml collagenase II , 1 mg/ml Protease E  plus 50 units/ml DNase I  at 37 °C for 5 min. Separated glomeruli were washed three times, then placed in 2 ml digestion buffer and incubated for an additional 30 minutes at 37℃ with rotating. Globes were removed and dissected in PBS on ice. Retina tissues were dissociated with dissociation system containing 2 mg/ml collagenase  II  and 200 units/ml DNase I at 37℃ for 30 minutes on a rotator. The retinal cells were then sorted by magnetic bead separation using PE anti-mouse CD140b antibody and PE-anti PECAM1 antibody.
Library was performed according to the manufacter’s instructions (single cell 3’ v2 protocol, 10x Genomics). Briefly, cells were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM). The poly-A RNA from the cell lysate contained in every single GEM was retrotranscripted to cDNA, which contains an Ilumina R1 primer sequence, Unique Molecular Identifier (UMI) and the 10x Barcode. The pooled barcoded cDNA was then cleaned up with Silane DynaBeads, amplified by PCR and the apropiated sized fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction Ilumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added. RNA libraries for RNA-seq were prepared using SMARTER mRNA-Seq Library Prep Kit following manufacturer's protocols.

treatment_protocol: HMC and HRPC was induced by treatment of 100 µg/ml AGEs for 6 hours while 100 µg/ml BSA as control.

value: kidney

tag: age

value: 21 weeks old

value: wide-type


title: T. gondii VEG WT, Switch Conditions, Rep 2

source_name: replicating tachyzoites under different conditions

value: tachyzoites and/or bradyzoites

value: high pH, low C02, low serum conditions


title: T. gondii VEG WT, Switch Conditions, Rep 1

value: replicating tachyzoites under different conditions


title: FACS-sorted cKIT(+) spermatogonia, Cbx2[+/+], rep3


title: FACS-sorted cKIT(+) spermatogonia, Cbx2[23KRA/23KRA], rep3


title: FACS-sorted cKIT(+) spermatogonia, Cbx2[+/+], rep2


title: T. gondii in vitro bradyzoite induction conditions 3


title: T. gondii VEG BFD1 KO, Control Conditions, Rep 3


title: Gfap_G_M_WT_F3

city: Los Angeles

first: Yuichiro

last: Itoh

state: California

address: 635 Charles E. Young Drive South

department: Neurology

institute: UCLA

description: astrocyte RNA isolated from GFAP-Cre:RiboTag mice

data_processing:
Data quality check was done on Illumina SAV. De-multiplexing was performed with the Illumina Bcl2fastq2 v 2.17 program.
Qualities of raw sequence data were examined using FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/), and Trimmomatic (Bolger et al., 2014) was used for cleaning.
R package “QuasR” (Gaidatzis et al., 2015) was used for the read alignment to the mouse genome (mm10) followed by counting at the gene level.
Assembly: mm10
Supplementary files format and content: Excel file includes raw counts

source_name: hippocampus

extract_protocol:
RNAs were isolated using the Direct-zol™ RNA MiniPrep Plus kit (Zymo Research).
The RNA sequencing library was made using KAPA Stranded RNA-Seq Kit (Kapa Biosystems) which consists of mRNA enrichment, cDNA generation, end repair, A-tailing, adaptor ligation, strand selection, and PCR amplification.

value: hippocampus

tag: Sex

value: female

value: astrocyte

value: wildtype

tag: gonad

value: GDX

value: 12-14 months


title: Gfap_S_M_WT_F1

value: sham


title: Gfap_S_M_bKO_F1

value: ERBeta cKO


title: Gfap_G_M_WT_F2


title: Gfap_G_M_WT_F4


title: Gfap_G_M_WT_F1


title: FACS-sorted cKIT(+) spermatogonia, Cbx2[23KRA/23KRA], rep4

value: Testes

value: cKIT(+) differentiating spermatogonia


title: FACS-sorted cKIT(+) spermatogonia, Cbx2[+/+], rep1


title: FACS-sorted cKIT(+) spermatogonia, Cbx2[23KRA/23KRA], rep1


title: Gfap_S_M_bKO_F3


title: Gfap_S_M_bKO_F4


title: Gfap_S_M_WT_F4


title: T. gondii in vitro control conditions 1

value: freshly excysted sporozoites

value: sporozoites grown in vitro

value: VEG

value: control media conditions


title: Gfap_S_M_WT_F3


title: FACS-sorted cKIT(+) spermatogonia, Cbx2[23KRA/23KRA], rep2


title: Gfap_S_M_bKO_F5


title: HeLa cells, shSAT1 #1

city: Tokyo

first: Ryuichi

last: Nakahara

address: 4-6-1 Komaba, Meguro

department: the Research Center of Advanced Science and Technology

country: Japan

institute: the University of Tokyo

data_processing:
Raw reads were trimmed using trim_galore(v0.5.0) with default parameters.
Reads were mapped to the genome (hg38) using STAR(v2.7.10)  with the parameters --outFilterMultimapNmax 1.
Unmap reads were then mapped to the mouse genome (mm10) using same parameters.
UMI counting was done using HTSeq-count(v2.0.2).
Gene expression levels were calculated and normalized using DESeq2(v2.1.34) with default parameters.
Assembly: hg38/mm10
Supplementary files format and content: tab-delimited text files include raw count values for each Sample
Supplementary files format and content: ded

organism: Homo sapiens

source_name: xenograft host: female scid/scid mice (C.B-17/Icr-scid/scidJcl) tissue type: snapfrozen xenograft specimen

growth_protocol: HeLa cells were maintained in DMEM supplemented with 10% fetal bovine serum (FBS) in humidified atmosphere with 5% CO2 at 37C.

extract_protocol: Total RNA from xenograft samples was extracted using the RNeasy Mini Kit (Qiagen), with a DNase incubation step included to ensure complete removal of genomic DNA.

treatment_protocol: 1x10^7 cells of HeLa were injected to scid/scid mice.Sacrifised 21 days after transplantation, tumour removed and frozen.

value: xenograft host:female scid/scid mice (C.B-17/Icr-scid/scidJcl) tissue type:snapfrozen xenograft specimen

tag: cell line

value: HeLa

value: carcinoma of cervix

value: SAT1 knockdown


title: HeLa cells, shControl #3

value: WT


title: HeLa cells, shControl #1


title: HeLa cells, shSAT1 #2


title: HeLa cells, shSAT1 #3


title: HeLa cells, shControl #2


title: THP-1-H-si2

data_processing:
Transcript abundances were quantified against the human reference genome (GRCh38) using Kallisto(v0.46.2).
Transcript counts were combined per gene to generate gene counts.
Downstream analysis was performed uging Kallisto normalized counts.
Assembly: hg38
Supplementary files format and content: tab-delimited text files include normalized values for each Sample

source_name: Blood

growth_protocol: THP-1 monocyte cells were maintained in RPMI1640 with 10% fetal bovine serum in humidified atmosphere with 5% CO2 at 37 ˚C

extract_protocol:
RNA was extracted using Rneasy extraction kit (Qiagen). 1.3ug of total RNA was used for the construction of libraries.
RNA libraries for RNA-seq were prepared using SMARTER mRNA-seq Library Prep Kit following manufacturer's protocols.

treatment_protocol: THP-1 cells were transfected with siRNA negative control (sc) or 2 kind of siRNAs targeting SREBP2 (si1/si2) for 48 hours. After transfection, cells were cultured in either control or hypoxic (1%O2) condition for 24 hours before extraction of total RNA.

value: Blood

value: Monocyte

value: SREBP2 KD

value: Hypoxia 24 hours


title: THP-1-C-siC

value: Control


title: THP-1-H-si1


title: THP-1-H-siC


title: CH04 Amplicon

city: New York

first: Dan

middle: A.

last: Landau

address: 101 6th Ave

department: Molecular Pharmacology Program

institute: New York Genome Center

description: 10x Genomics

data_processing:
10X Illumina data was processed using Cell Ranger (v.3.1.0) with default parameters and reads were aligned to the human reference sequence. CITE-seq samples were processed in a similar way to the described in the ADT manual from 10x Genomics.
Genotyping of single cells was carried out with the IronThrone (v.2.1) pipeline. In brief, individual amplicon reads were assessed for the appropriate structure (i.e. presence of the primer sequence and the expected sequence between the primer and given mutation site) and all reads were assessed for a matching cell barcode to the list generated from the 10X paired GEX dataset. A Levenshtein distance of 0.1 was allowed for all sequence matching and collapsing steps and only UMIs with a minimum of 2 supporting reads were retained for final genotyping. Following UMI collapse, genotype assignment of individual UMIs was conducted as described previously with majority rule of supporting reads for wildtype or mutant status (using a 0.7 PCR read ratio, above which the majority of PCR reads must be in order for a UMI to be called definitively). Rare UMIs that did not pass this threshold were removed as ambiguous. Additionally, to remove reads that result from PCR recombination, UMIs in the amplicon library that match UMIs of non-SF3B1 genes in the gene expression library were discarded. Each single cell was assigned as either mutant or wildtype as follows: cells with at least 1 mutant UMI were assigned as mutant cells and cells with 0 mutant UMIs and at least 1 wildtype UMI were assigned as wildtype.
scRNA-seq ONT long-read sequencing data processing, alignment, junction calling and annotation
Guppy v3.0.6 - 4.0.11 was used for basecalling output from ONT sequencing to create FAST5 files that were then converted to FASTQ files. After generating FASTQ files from the long-read ONT sequencing, we then filtered for only reads containing a polyA tail within 100 base pairs of either 5’ or 3’ end using the `NanoporeReadScanner-0.5.jar` within the SiCeLoRe-1.0 workflow. Filtered reads are aligned to the primary human genome, assembly GRCh38.p12 using minimap2 (v.2.17). Minimap2 was used with the `-ax splice` flag to prioritize annotated splice junctions. Additionally we made use of the `--junc-bed` option, to increase alignment scores for those splice junctions found in the reference junction bed file. For our reference junctions, we used splice junctions from single-cell SMART-seq2 data from human CD34+ cells obtained from a CH sample with no SF3B1 mutation. Additionally we used `--secondary=no` to suppress multi-mappings.
In preparation to identify the cell barcodes and UMI’s present in the long-read sequencing, we used the `IlluminaParser-1.0.jar` in SiCeLoRe to parse the cell barcodes and UMI’s present in the complementary short-read sequencing library. We continued to use SiCeLoRe to tag the aligned bam files with cell barcodes and UMI’s identified in the short-read library, and generate consensus sequences for each unique cell barcode and UMI combination. Consensus sequences were used to create a gene by cell count matrix.Intron-junction calling is then performed on consensus sequence BAM files, adapted from the method used in the LeafCutter pipeline for short read RNA-seq data. In brief, the Intron junction calling pipeline utilizes the pysam.fetch() function and iterates through each transcript in the bam file, noting its cell barcode (CB) tag as well as the coordinates of each intron-junction for that transcript. On iterating through the bam file, counts for the usage of each unique intron-junction and the corresponding CB are recorded. This ultimately generates an Intron-Junction x Cell Barcode count matrix for the given bam file. Each intron-junction is then identified using annotations available in the GENCODE GRCh38.p12 v31 basic annotation reference file as either canonical 3’, canonical 5’, alternative 3’, alternative 5’. This outputs a metadata file with annotations for each junction corresponding to the junctions of the Intron-Junction x Cell Barcode count matrix.
Assembly: hg38
Supplementary files format and content: Processed 10X data is matrix table of  gene counts per cell for each sample
Supplementary files format and content: Output matrices processed from amplicon data have five columns: number of wildtype calls (WT.calls),  number of mutant calls (MUT.calls) , genotype (Genotype), cell assignments (Cell.Assignment). Row names are the corresponding barcodes..
Supplementary files format and content: Junction files contain a junction (rows) per cell barcode (columns) matrix of read counts. Similar structure for exon counts
Supplementary files format and content: ADT cell ranger output (barcodes, matrix features)

source_name: PBMC

extract_protocol:
Cryopreserved mononuclear cells isolated from bone marrow biopsies or peripheral blood from myelodysplastic syndrome patients with SF3B1 mutations were retrieved from Memorial Sloan Kettering, and University of Manchester. Additionally, cryopreserved G-CSF mobilized stem cell grafts (without additional mobilizing agents such as plerixafor or cyclophosphamide) from CH patients with SF3B1 mutations were retrieved from the Dana Farber Cancer Institute. Cryopreserved mononuclear cells and grafts were thawed and stained using standard procedures. Cells were first incubated with Human FcX blocking solution (Biolegend, #422302) and then incubated with the surface antibody CD34-PE-Vio770 (clone AC136, lot #5180718070, dilution 1:50, Miltenyi Biotec) and DAPI (Sigma-Aldrich) for 10 minutes at 4°C. Cells were then sorted for DAPI-negative, CD34+ cells using BD Influx at the Weill Cornell Medicine flow cytometry core. Additionally, cryopreserved G-CSF mobilized stem cell grafts (without additional mobilizing agents such as plerixafor or cyclophosphamide) from CH patients with SF3B1 mutations were retrieved from the Dana Farber Cancer Institute and the Weizmann Institute of Science. To confirm the absence of additional genetic mutations, CH samples were sequenced using a previously described panel135 that includes myeloma driver mutations as well as CH-specific mutations. All samples underwent ultra-low pass whole genome sequencing, rejecting the presence of tumor contamination. Cryopreserved mononuclear cells and grafts were thawed and stained using standard procedures. Cells were first incubated with Human FcX blocking solution (Biolegend, #422302) and then incubated with the surface antibody CD34-PE-Vio770 (clone AC136, lot #5180718070, dilution 1:50, Miltenyi Biotec) and DAPI (Sigma-Aldrich) for 10 minutes at 4°C. Cells were then sorted for DAPI-negative, CD34+ cells using BD Influx at the Weill Cornell Medicine flow cytometry core.
The standard 10x Genomics Chromium 3’ (v.3.1 chemistry) and CITE-seq protocols35,36 were carried out according to manufacturer’s recommendations for the generation of scRNA-seq and ADT libraries (Fig. 1A). At the cDNA amplification step in the 10x Genomics protocol, 1 μL of 1 μM spike-in primer (5’ –GATCCTCGTCCTCATTGAACCGC– 3’) was added to increase the yield of SF3B1 cDNA and 1 µL of 0.2 µM ADT PCR additive primer (5’ – CCTTGGCACCCGAGAATTCC – 3’) was added to amplify ADT. After cDNA amplification and a double-sided cleanup with SPRI beads to separate cDNA and ADT fractions, the ADT fraction was amplified for 10 cycles with SI-PCR oligo (10x Genomics) and TruSeq Small RNA RPI-x (Illumina) primers to index the samples. SPRI was used to clean up the ADT final products.In both samples in which CITE-seq was conducted and not conducted, cDNA was allocated for gene expression library creation (standard 10x protocol; 25% of cDNA), targeted genotyping (10% of cDNA), and ONT on-bead library preparation (10ng of cDNA). Any remaining cDNA was stored.For locus-specific amplification (GoT), two serial PCRs were performed with nested reverse primers, based on the SF3B1 mutation of interest. For mutations upstream of K700E, (5’ – GATCCTCGTGGTCATTGAACCGC – 3’ and 5’ – CACCCGAGAATTCCAGGCTACTATGATCTCTACCATGAGACCTG – 3’) and, for K700E mutations, (5’ – GTGCAAAAGCAAGAAGTCCT – 3’ and 5’ –CACCCGAGAATTCCATGAACATGGTCTTGTGGATGAGC – 3’) were used as reverse primers. These reverse primers and the generic forward SI-PCR amplify the site of interest from the cDNA template (10 PCR cycles each). The second locus-specific reverse primers contain a partial Illumina TruSeq Small RNA read 2 handle and a locus-specific region to allow SF3B1 specific priming. The SI-PCR oligo (10x Genomics) anneals to the partial Illumina TruSeq read 1 sequence, preserving the cell barcode (CB) and unique molecule identifier (UMI). After these rounds of amplification and SPRI purification to remove unincorporated primers, a third PCR was performed with a generic forward PCR primer (P5_generic, 5’ – AATGATACGGCGACCACCGAGATCTACAC – 3’) to retain the CB and UMI together with an RPI-x primer (Illumina) to complete the P7 end of the library and add a sample index (6 PCR cycles).Gene expression, ADT, and SF3B1 amplicon libraries were pooled to receive 25,000, 5,000, and 5,000 reads per cell, respectively, during Illumina sequencing. The cycle settings were as follows: 28 cycles for read 1, 90 cycles for read 2, 10 cycles for i7, and 10 cycles for i5 sample index. To examine splicing patterns broadly in the whole transcriptome, full length cDNA was sequenced using the Oxford Nanopore Technology sequencing platform on PromethION flow cells. To enrich for transcripts that contain CBs and UMIs and decrease the presence of PCR artifacts, on-bead PCR with a biotinylated primer selecting for an adapter upstream of the CB was completed. In brief, 10ng of full length cDNA was amplified with LongAmp master mix (NEB) and TSO (5’ – NNNAAGCAGTGGTATCAACGCAGAG – 3’) and biotinylated read 1 (5’ – /5Biosg/AAAAACTACACGACGCTCTTCCGATCT – 3’) primers for 5 cycles. After cleanup with SPRI, 1X and 5X SSPE-washed M270 streptavidin beads (ThermoFisher) were added to the PCR product. After a 15 minute incubation and washes with 1X SSPE and 10mM Tris-HCl (pH 8, the bead-bound PCR products and beads were resuspended in PCR master mix for further amplification. Samples were amplified with LongAmp master mix and TSO and read 1 (5’ – NNNCTACACGACGCTCTTCCGATCT – 3’) primers for 5 cycles. After cleanup with SPRI, 1000ng of each full length cDNA library was sequenced on one the PromethION flow cell.
Single-cell RNA-seq (10X and long-read ONT) with targeted amplification of a mutation of interest. Two runs of the experiment, A and B, were completed for sample MDS02.

value: PBMC

tag: disease

value: Clonal Hematopoiesis


title: CH03 Amplicon

description: 10x Genomics


title: CH04 10X

description: 10x Genomics


title: AML01B 10X

description: 10x Genomics

value: Acute Myeloid Leukemia


title: AML01A 10X

description: 10x Genomics


title: MDS02B ADT

description:
10x Genomics
TotalSeq-C- Universal Human panel + CD34 and CD90 (BioLegend)

source_name: Bone marrow

value: Bone marrow

value: Myelodysplatic syndrome


title: CH03 10X

description: 10x Genomics


title: MDS03 ADT


title: MDS02A ADT


title: 21_WD+CHP_rep5

city: Lausanne

first: Keno

last: Strotjohann

address: Route Cantonale

department: School of Life Sciences, Institute of Bioengineering

country: Switzerland

institute: École Polytechnique Fédérale de Lausanne (EPFL)

data_processing:
After sequencing on the BGIseq-500 platform, the raw reads were filtered. Data filtering includes removing adaptor sequences, contamination and low-quality (phred quality < 20) reads from raw reads.
Sequenced reads were mapped to the GRCm38   mm10   primary   assembly  of the mouse genome using ensembl release 95 gene annotation with STAR aligner version 2.7.3a
Reads were counted using STAR aligner
Assembly: mm10
Supplementary files format and content: csv file with raw counts for every gene and sample

source_name: WD+CHP

extract_protocol:
RNA was extracted in Trizol (TriPure, Roche) from frozen liver tissue, using the Direct-zol-96 RNA Kit (Zymo Research).
RNA libraries were prepared for sequencing using standard BGISeq500 protocols with polyA enrichment

value: mouse kidney

value: WD+CHP


title: HL60_COMBO_3

first: Tomohiro

last: Yabushita

address: 7-3-1, Hongou, Bunkyo-ku

institute: The University of Tokyo

data_processing:
Read quality was confirmed using FastQC.
Sequence reads were trimmed for adaptor sequence/low-quality sequence.
These reads were aligned to the GRCh38 reference genome using the HISAT2 pipeline.
Transcripts per million (TPM) were calculated based on the length of the gene and the number of reads mapped to each gene.
Differential expression analysis among each group was performed using the DESeq2 R package.
Assembly: GRCh38
Supplementary files format and content: tab-delimited text file includes raw counts for each sample.
Supplementary files format and content: tab-delimited text file includes TPM counts for each sample.

source_name: hematopoetic

growth_protocol: HL-60 (CCL-240, ATCC) cells were maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% P/S.

extract_protocol:
Total RNA was extracted using an RNeasy Mini Kit (Qiagen), and RNA integrity and purity were checked using Agilent 2100 Bioanalyzer (Agilent Technologies). 1.0μg of total RNA was used for the construction of sequencing libraries.
After mRNA enrichment using oligo(dT) beads and rRNA depletion using the Ribo-Zero Plus rRNA Depletion Kit (Illumina), cDNA was synthesized using an mRNA template and random primers, second-strand synthesis buffer, dNTPs, RNase H and DNA polymerase I. The double-stranded cDNA library was completed by “A” base ligation and sequencing adaptor ligation. The quality and quantity of these libraries were checked using Qubit 2.0 (Thermo Fisher Scientific), Agilent 2100, and quantitative PCR using the Real-time PCR Systems Step One Plus (Applied Biosystems).

treatment_protocol: HL-60 cells in each group (DMSO, DAC 0.5 μM, simvastatin 10 μM, and DAC+simvastatin) were harvested 36 hours after exposure to each treatment

value: hematopoetic

value: non-adherent

value: Decitabine+Simvastatin


title: 19_WD+CHP_rep4


title: HL60_COMBO_2


title: 18_WD+CHP_rep3


title: HL60_SIMVA_3

value: Simvastatin


title: 16_WD+CHP_rep1

value: WT


title: HL60_COMBO_1


title: 17_WD+CHP_rep2


title: Case No. 1

type: genomic

city: Changchun

first: Hongguo

last: Zhang

state: Jilin

address: 1 Xinmin Street, Changchun, Jilin 130021, P.R. China

department: Center for Reproductive Medicine and Center for Prenatal Diagnosis

institute: First Hospital, Jilin University, Changchun, China

description: Increased nuchal translucency (4.5mm)

hyb_protocol: DNA was restriction digested, PCR amplified, fragmented, labeled and hybridized to each array according to the manufacturer's instructions.

scan_protocol: The arrays were then washed using Affymetrix Fluidics Stations, and scanned using the GeneChip System 3000Dx v.2.

data_processing: ChAS console from Affymetrix was used to assess genome wide copy number frequencies. Log2 Ratio copy number values with associated chromosome positions are available on the series record.

label: biotin

molecule: genomic DNA

source_name: amniotic fluid fetal cells

label_protocol: Labelling and hybridizations were performed according to the manufacturer’s protocol.

growth_protocol: not applicable

extract_protocol: Genomic DNA of amniotic fluid cells or peripheral blood sampling of the parents was extracted with using the QIAamp DNA Blood Mini Kit (Qiagen, Inc., Hilden, Germany) according to the manufacturer’s protocol.

treatment_protocol: not applicable

value: amniotic fluid fetal cells

tag: diagnosis

value: microdeletion of 1q21.1


title: Case No. 25

description: ,developmental delay and scoliosis)


title: HL60_SIMVA_2


title: Case No. 2

description: Wife:1q21.1 deletion carrier


title: 13_WD+water_rep5

source_name: WD+water

value: WD+water


title: HL60_SIMVA_1


title: Case No. 5

description: Non-invasive prenatal testing infers high risk of chromosome 9


title: 12_WD+water_rep4


title: HL60_DAC_3

value: Decitabine


title: 10_WD+water_rep3


title: Case No. 23

description: Increased nuchal translucency


title: Case No. 6

description: Aberrant right subclavian artery; ventricular apical thin point


title: Case No. 12

description: advanced maternal age


title: HL60_DAC_2


title: Case No. 26

description: advanced maternal age, tetralogy of fallot


title: 9_WD+water_rep2


title: HL60_DAC_1


title: Case No. 19

description: Voluntary request, no abnormal ultrasound findings observed


title: 8_WD+water_rep1


title: HL60_DMSO_3

value: DMSO


title: Case No. 14

description: advanced maternal age, prenatal ultrasound findings infer cerebral ventriculomegaly


title: 7_CD+water_rep5

source_name: CD+water

value: CD+water


title: Case No. 11

description: NIPT infers high risk of chromosome 1


title: Case No. 3

description: Non-invasive prenatal testing infers high risk of chromosome 16; fetal growth restriction and microcephaly(35w6d)


title: Case No. 22

description: advanced maternal age


title: Case No. 15

description: abnormal childbearing history (child presenting language retardation）


title: Case No. 4

description: Increased nuchal translucency(3.1mm), abnormal childbearing history (boy: developmentally delay and intellectual disability presenting 46,XY,t(1;6)(p22;q21) and 1q21.1 microdeletion


title: HL60_DMSO_2


title: 5_CD+water_rep4


title: Case No. 17

description: Prenatal ultrasound findings infers ventricular septal defect and absence of nasal bone


title: HL60_DMSO_1


title: 2_CD+water_rep2


title: Case No. 9

description: Abnormal childbearing history (a fetus presenting VSD with maternally inherited 1q21.1 deletion (TOP at 37w)


title: Case No. 8

description: advanced maternal age


title: 1_CD+water_rep1


title: 3_CD+water_rep3


title: Case No. 10

description: advanced maternal age, increased nuchal translucency


title: Case No. 21

description: Husband: 46,XY,inv(6)(p21.1q25)


title: Case No. 13

description: abnormal childbearing history (child with cardiac malformation）


title: 40: WD+CHP_rep3

source_name: mouse gastrocnemius

extract_protocol:
RNA was extracted in Trizol (TriPure, Roche) from frozen liver tissue, using the NucleoSpin RNA Kit (Macherey-Nagel).
RNA libraries were prepared for sequencing using standard BGISeq500 protocols with polyA enrichment

value: mouse gastrocnemius

value: male


title: Case No. 24

description: advanced maternal age, abnormal childbearing history (child presenting cerebral palsy


title: 42: WD+CHP_rep5


title: Case No. 16

description: Increased nuchal translucency, absence of nasal bone


title: 41: WD+CHP_rep4


title: 38: WD+CHP_rep1


title: 35: WD+water_rep5


title: exp4_ch11_TCR

city: Oxford

first: Lucy

last: Garner

address: Experimental Medicine, Level 5, John Radcliffe Hospital

department: Nuffield Department of Medicine

country: United Kingdom

institute: University of Oxford

description: 10x Genomics

library_source: other

data_processing:
FASTQ files for all modalities (GEX, ADT/HTO, TCR) were processed using the Cell Ranger (v7.0.1) multi pipeline.
Assembly: GRCh38
Supplementary files format and content: Cell Ranger output files: barcodes.tsv.gz, features.tsv.gz, matrix.mtx.gz (GEX, ADT, HTO); filtered_contig_annotations.csv (TCR)
Supplementary files format and content: TotalSeq_C_Human_Universal_Cocktail_v1_137_Antibodies_399905_Barcodes.xlsx (TotalSeq-C Human Universal Cocktail antibodies and barcodes)
Library strategy: scTCR-seq

molecule: other

source_name: Channel 11; donors 4.1, 4.2, 4.3; unstim, TCR+cytokine; 68h stimulation; TCR repertoire sequencing

extract_protocol:
Isolated blood T cells were left unstimulated or stimulated for 20 h with MR1/5-OP-RU (TCR), IL-12 + IL-18 (cytokine) or both, before MAIT cell (CD3+CD26+CD161hiVα7.2+) sorting. Additionally, isolated T cells were left unstimulated or stimulated for 68 h with both MR1/5-OP-RU and IL-12 + IL-18 (TCR+cytokine), before MAIT cell sorting. Single-cell gene, surface protein and TCR repertoire analysis was performed using 10x Genomics technology.
Sequencing libraries were generated using the 10x Genomics Chromium Next GEM Single Cell 5' Reagent Kits v2 (Dual Index).

tag: donor

value: Blood

value: MAIT cells

value: unstim, TCR+cytokine

tag: time

value: 68 h stimulation

tag: library type

value: TCR


title: exp4_ch12_TCR

description: 10x Genomics

source_name: Channel 12; donors 4.1, 4.2, 4.3; unstim, TCR+cytokine; 68h stimulation; TCR repertoire sequencing


title: exp4_ch10_TCR

description: 10x Genomics

source_name: Channel 10; donors 4.1, 4.2, 4.3; unstim, TCR+cytokine; 68h stimulation; TCR; TCR repertoire sequencing


title: Case No. 20

description: prenatal ultrasound findings infer cerebral ventriculomegaly


title: Case No. 18

description: advanced maternal age, abnormal childbearing history (trisomy 21)


title: 39: WD+CHP_rep2

value: WD+CHP


title: exp4_ch9_TCR

description: 10x Genomics

source_name: Channel 9; donors 4.1, 4.2, 4.3; unstim, TCR+cytokine; 68h stimulation; TCR repertoire sequencing


title: 34: WD+water_rep4


title: exp4_ch8_TCR

description: 10x Genomics

source_name: Channel 8; donors 4.1, 4.2, 4.3; unstim, TCR, cytokine, TCR+cytokine; 20h stimulation; TCR repertoire sequencing

value: unstim, TCR, cytokine, TCR+cytokine

value: 20 h stimulation


title: exp4_ch7_TCR

description: 10x Genomics

source_name: Channel 7; donors 4.1, 4.2, 4.3; unstim, TCR, cytokine, TCR+cytokine; 20h stimulation; TCR repertoire sequencing


title: AML01B ONT

description: ONT

value: PBMC

value: Acute Myeloid Leukemia


title: 31: WD+water_rep3

value: WT


title: exp4_ch6_TCR

description: 10x Genomics

source_name: Channel 6; donors 4.1, 4.2, 4.3; unstim, TCR, cytokine, TCR+cytokine; 20h stimulation; TCR repertoire sequencing


title: 30: WD+water_rep2

value: WD+water


title: 29: WD+water_rep1


title: AML01A ONT

description: ONT


title: 28: CD+water_rep5


title: exp4_ch5_TCR

description: 10x Genomics

source_name: Channel 5; donors 4.1, 4.2, 4.3; unstim, TCR, cytokine, TCR+cytokine; 20h stimulation; TCR repertoire sequencing


title: CH03 ONT-junctions

description: ONT

value: Clonal Hematopoiesis


title: nup1-1 5d ABA Rep3

city: London

first: Yuhai

last: Cui

state: ON

address: 1391 Sandford St,

country: Canada

institute: Agriculture and AgriFood Canada

description: mRNA

data_processing:
Quality of raw reads were checked by FastQC.
Trimmomatic was used to trim the ends.
Tophat was used for alignment with reference genome.
Cufflink package was used to identify differentially expressed genes.
Genome_build: TAIR10 Arabidopsis
Supplementary_files_format_and_content: *.txt: Tab-delimited text files showing differentially expressed genes.

organism: Arabidopsis thaliana

source_name: seedlings

growth_protocol: Arabidopsis were grown on MS media for 5 days.

extract_protocol:
mRNA from young seedlings was isolated using RNeasy Plant Mini Kit (Qiagen) following the manufacturer’s protocol.
RNA libraries were prepared for sequencing using standard Illumina protocols.

treatment_protocol: Seedlings were grown in ABA supplemented MS media.

value: Seedlings

value: 5 days

value: ABA


title: exp4_ch4_TCR

description: 10x Genomics

source_name: Channel 4; donors 4.1, 4.2, 4.3; unstim, TCR, cytokine, TCR+cytokine; 20h stimulation; TCR repertoire sequencing


title: 27: CD+water_rep4

value: mouse gastrocnemius

value: CD+water

value: male


title: nup1-1 5d ABA Rep2

description: mRNA


title: exp4_ch3_TCR

description: 10x Genomics

source_name: Channel 3; donors 4.1, 4.2, 4.3; unstim, TCR, cytokine, TCR+cytokine; 20h stimulation; TCR repertoire sequencing

value: Blood

value: MAIT cells


title: AML01B Amplicon

description: ONT

source_name: PBMC


title: 25: CD+water_rep3


title: nup1-1 5d ABA Rep1

description: mRNA


title: nup1-1 5d Rep2

description: mRNA


title: exp4_ch2_TCR

description: 10x Genomics

source_name: Channel 2; donors 4.1, 4.2, 4.3; unstim, TCR, cytokine, TCR+cytokine; 20h stimulation; TCR repertoire sequencing

value: TCR


title: 24: CD+water_rep2


title: nup1-1 5d Rep 3

description: mRNA


title: nup1-1 5d Rep1

description: mRNA


title: AML01A Amplicon

description: ONT


title: 22: CD+water_rep1


title: Case No. 7

description: Risk of fetal trisomy 21: 1/53

source_name: amniotic fluid fetal cells

value: amniotic fluid fetal cells

value: microdeletion of 1q21.1


title: exp4_ch1_TCR

description: 10x Genomics

source_name: Channel 1; donors 4.1, 4.2, 4.3; unstim, TCR, cytokine, TCR+cytokine; 20h stimulation; TCR repertoire sequencing

value: unstim, TCR, cytokine, TCR+cytokine

value: 20 h stimulation


title: exp4_ch12_ADT_HTO

description:
10x Genomics
Processed data available on GEX samples.

data_processing:
FASTQ files for all modalities (GEX, ADT/HTO, TCR) were processed using the Cell Ranger (v7.0.1) multi pipeline.
Assembly: GRCh38
Supplementary files format and content: Cell Ranger output files: barcodes.tsv.gz, features.tsv.gz, matrix.mtx.gz (GEX, ADT, HTO); filtered_contig_annotations.csv (TCR)
Supplementary files format and content: TotalSeq_C_Human_Universal_Cocktail_v1_137_Antibodies_399905_Barcodes.xlsx (TotalSeq-C Human Universal Cocktail antibodies and barcodes)
Library strategy: CITE-seq

molecule: protein

source_name: Channel 12; donors 4.1, 4.2, 4.3; unstim, TCR+cytokine; 68h stimulation; ADT + HTO

value: unstim, TCR+cytokine

value: 68 h stimulation

value: ADT + HTO

tag: adt

value: TotalSeq-C0251 anti-human Hashtag 1 Antibody, TotalSeq-C0252 anti-human Hashtag 2 Antibody, TotalSeq-C0253 anti-human Hashtag 3 Antibody, TotalSeq-C0254 anti-human Hashtag 4 Antibody, TotalSeq-C0256 anti-human Hashtag 6 Antibody, TotalSeq-C0257 anti-human Hashtag 7 Antibody


title: CH04 ONT-junctions

description: ONT

value: PBMC

value: Clonal Hematopoiesis


title: Col-0 5d ABA Rep 3

description: mRNA

value: Seedlings

value: 5 days

value: ABA


title: exp4_ch11_ADT_HTO

source_name: Channel 11; donors 4.1, 4.2, 4.3; unstim, TCR+cytokine; 68h stimulation; ADT + HTO


title: Col-0 5d ABA Rep2

description: mRNA


title: CCl4+CHP_rep4

first: Xiaoxu

last: Li

address: Station 15 - CH

institute: EPFL

data_processing:
After sequencing, the raw reads were filtered. Data filtering includes removing adaptor sequences, contamination and low-quality (phred quality < 20) reads from raw reads.
Sequenced reads were mapped to the GRCm38   mm10   primary   assembly  of the mouse genome using ensembl release 102 gene annotation with STAR aligner version 2.7.3a
Reads were counted using STAR aligner
Assembly: Mus_musculus.GRCm38.102
Supplementary files format and content: xlsx file with counts per gene per sample

source_name: mouse liver

extract_protocol:
Total RNA was extracted from the liver using NucleoZOL reagent (740404.200, Macherey-Nagel).
RNA libraries were prepared for sequencing using Illumina TruSeq Stranded Total RNA Library Prep Gold Kit

value: mouse liver

value: WT


title: CCl4+CHP_rep5


title: exp4_ch10_ADT_HTO

source_name: Channel 10; donors 4.1, 4.2, 4.3; unstim, TCR+cytokine; 68h stimulation; ADT + HTO

value: Blood

value: MAIT cells


title: FACS-sorted cKIT(+) spermatogonia, Cbx2 amplicon, WT band, Rosa26-CreERT2; Cbx2[Flox/Flox], rep4

description:
Cbx2 amplicon generated using cell-barcoded cDNA
Cbx2_iKO_amplicon_WTBand_rep4

data_processing:
Cellranger count (cellranger/6.0) was used to map fastq files to the mouse genome (refdata-cellranger-mm10-3.0.0) and to obtain gene expression counts per cell. Cells were filtered by a) nFeature_RNA > 2000, b) mitochondrial RNA percentage < 7.5, c) percent occupied by the largest gene < 3, and d) RNA count and feature ratio < 5.5 (see scripts posted on https://github.com/jongminkmg/Cbx2). Four control and four inducible KO samples were integrated to align cell populations across all eight samples. Two control samples each generated from different mouse litters and batches of reagents were used as initial references for integration. Integrated data were then scaled and processed with 20 principal components for clustering. Differential gene expression tests were performed by the FindMarkers function of Seurat using Wilcoxon rank sum test.
Cbx2 genotypes were assigned based on independent Cbx2 amplicon sequencing data using a custom Python script (https://github.com/jongminkmg/Cbx2).  The script identifies 'TGGCGCATCTGGTTCCTTGAGCTTGGAGCG' (WT, exon5 linked to exon4) or 'TGGCGCATCTGGTTCTTGGAGGACCAGCCG' (Mut, exon5 linked to exon2) from the read2 of the paired-end reads, and assigns the genotypes to the associated read1 (UMI-barcode). When an UMI is assigned to two different genotypes, likely due to PCR chimera formation, the genotype was left unassigned unless reads supporting one genotype are present 10 times more than the other genotype. A cell was assigned to be 'Mut' when all Cbx2 amplicon reads show recombination (exon5 linked to exon2), and otherwise assigned to be 'WTorHet'. Only the cells that have at least four UMIs linked to Cbx2 sequence were assigned genotypes. The metadata for their genotypes (WT/Het or Mut) were added to the Seurat object by the AddMetaData function.
Supplementary files format and content: Tab-separated values files and matrix files

source_name: Testes

extract_protocol:
c-KIT(+) spermatogonia were FACS-sorted to concentration of  about 1000 cells/µL, and a total ~8000 cells were loaded into the Chromium controller. We used 5' barcoding instead of 3' barcoding because deleted exons of Cbx2 were close to the 5' end of the gene. Single cell analysis with targeted genotyping was done by adapting 'Genotyping of Transcriptome (GoT)' (Nam et al., 2019, PMID: 31270458) and V(D)J region amplification of T or B-cell receptor protocol by the 10X Genomics. We aimed to make specific Cbx2 amplicons from cell-barcoded cDNA to amplify cDNA fragments and sequence Exon5 junctions to distinguish wild type (Exon4 spliced to Exon5) and mutant (Exon2 spliced to Exon5 after the deletion of Exon3, 4) for specific cell barcodes.
Cbx2 inducible knock out & FACS-sorted cKIT(+) spermatogonia profiling : single cell barcoded cDNA was made with Chromium Next GEM Single Cell 5' Reagent Kits (10X Genomics, Cat#:1000265) based on the manufacturer's protocol with 14 PCR cycles of cDNA amplification. 50 ng of the amplified cDNA based on the manufacturer’s protocol. 14 cycles of library amplification indexing PCR was performed. To generate Cbx2 amplicons, we performed nested PCRs to amplify Cbx2-specific cDNAs with cell barcodes. In the second PCR step of the nested PCR, Cbx2-specific primer that has a partial Illumina read2 sequence was used. The Cbx2-specific second nested primer also contains 'stagger' sequences between the Cbx2-specific region and partial read2 to increase library complexity for accurate base calls during Illumina sequencing. We confirmed the amplification of the correct-sized bands corresponding to wild-type and deleted Cbx2 amplicons by agarose gel electrophoresis. The amplified bands were excised and gel purified. Index PCR was performed to make the final Illumina-compatible libraries with unique indices with 10X provided index-PCR primers. Cbx2 amplicon libraries were pooled with standard 10X single cell gene expression libraries to occupy ~2% of total reads. A total of 4 samples were sequenced in one P3 lane on a NextSeq 2000 (NextSeq 2000 P3 Reagents (100 Cycles) Cat:20040559).

treatment_protocol: Tamoxifen (Sigma-Aldrich, Cat# T5648) was dissolved in corn oil (Spectrum Chemical, Cat# CO136) to a final 20 mg/mL by incubating the mixture at 37 ºC overnight while rotating. Dissolved tamoxifen was filtered and aliquots for daily use were stored in sterile tubes and kept at 4 ºC for up to 4 days. For the Cbx2 amplicon single cell RNA-seq experiments, 150 µL of warmed-up tamoxifen-corn oil was injected intraperitoneally daily for 3 consecutive days. No treatment was applied for profiling cells from the wild-type adult testis experiment.

value: Testes

value: cKIT(+) differentiating spermatogonia

value: Rosa26-CreERT2; Cbx2[Flox/Flox]

value: 150uL 20mg/mL Tamoxifen 3X IP injections


title: FACS-sorted cKIT(+) spermatogonia, Cbx2 amplicon, Mut band, Rosa26-CreERT2; Cbx2[Flox/Flox], rep4

description:
Cbx2 amplicon generated using cell-barcoded cDNA
Cbx2_iKO_amplicon_MutBand_rep4


title: Col-0 5d ABA Rep 1

description: mRNA


title: CCl4+CHP_rep3


title: FACS-sorted cKIT(+) spermatogonia, Cbx2 amplicon, Mut band, Rosa26-CreERT2; Cbx2[Flox/Flox], rep3

description:
Cbx2 amplicon generated using cell-barcoded cDNA
Cbx2_iKO_amplicon_MutBand_rep3


title: exp4_ch9_ADT_HTO

source_name: Channel 9; donors 4.1, 4.2, 4.3; unstim, TCR+cytokine; 68h stimulation; ADT + HTO

value: unstim, TCR+cytokine


title: Col-0 5d Rep2

description: mRNA


title: FACS-sorted cKIT(+) spermatogonia, Cbx2 amplicon, WT band, Rosa26-CreERT2; Cbx2[Flox/Flox], rep3

description:
Cbx2 amplicon generated using cell-barcoded cDNA
Cbx2_iKO_amplicon_WTBand_rep3


title: CCl4+CHP_rep1


title: exp4_ch8_ADT_HTO

source_name: Channel 8; donors 4.1, 4.2, 4.3; unstim, TCR, cytokine, TCR+cytokine; 20h stimulation; ADT + HTO

value: unstim, TCR, cytokine, TCR+cytokine

value: 20 h stimulation

value: TotalSeq-C0251 anti-human Hashtag 1 Antibody, TotalSeq-C0252 anti-human Hashtag 2 Antibody, TotalSeq-C0253 anti-human Hashtag 3 Antibody, TotalSeq-C0254 anti-human Hashtag 4 Antibody, TotalSeq-C0256 anti-human Hashtag 6 Antibody, TotalSeq-C0257 anti-human Hashtag 7 Antibody, TotalSeq-C0258 anti-human Hashtag 8 Antibody, TotalSeq-C0259 anti-human Hashtag 9 Antibody, TotalSeq-C0260 anti-human Hashtag 10 Antibody, TotalSeq-C0262 anti-human Hashtag 12 Antibody, TotalSeq-C0263 anti-human Hashtag 13 Antibody, TotalSeq-C0264 anti-human Hashtag 14 Antibody


title: Col-0 5d Rep1

description: mRNA


title: CCL4_rep5


title: FACS-sorted cKIT(+) spermatogonia, Rosa26-CreERT2; Cbx2[Flox/+] mock injected, rep4

description:
Used Chromium Next GEM Single Cell 5' Reagent Kit by 10X Genomics
Cbx2_WT_rep4

value: Rosa26-CreERT2; Cbx2[Flox/+]

value: 150uL mock corn oil 3X IP injections


title: exp4_ch7_ADT_HTO

source_name: Channel 7; donors 4.1, 4.2, 4.3; unstim, TCR, cytokine, TCR+cytokine; 20h stimulation; ADT + HTO

value: ADT + HTO


title: CCL4_rep4


title: exp4_ch6_ADT_HTO

source_name: Channel 6; donors 4.1, 4.2, 4.3; unstim, TCR, cytokine, TCR+cytokine; 20h stimulation; ADT + HTO


title: CCL4_rep3


title: FACS-sorted cKIT(+) spermatogonia, Cbx2[Flox/Flox], rep3

description:
Used Chromium Next GEM Single Cell 5' Reagent Kit by 10X Genomics
Cbx2_WT_rep3


title: exp4_ch5_ADT_HTO

source_name: Channel 5; donors 4.1, 4.2, 4.3; unstim, TCR, cytokine, TCR+cytokine; 20h stimulation; ADT + HTO


title: FACS-sorted cKIT(+) spermatogonia, Rosa26-CreERT2; Cbx2[Flox/Flox], rep4

description:
Used Chromium Next GEM Single Cell 5' Reagent Kit by 10X Genomics
Cbx2_iKO_rep4


title: exp4_ch4_ADT_HTO

source_name: Channel 4; donors 4.1, 4.2, 4.3; unstim, TCR, cytokine, TCR+cytokine; 20h stimulation; ADT + HTO


title: CCL4_rep2


title: FACS-sorted cKIT(+) spermatogonia, Rosa26-CreERT2; Cbx2[Flox/Flox], rep3

description:
Used Chromium Next GEM Single Cell 5' Reagent Kit by 10X Genomics
Cbx2_iKO_rep3


title: Col-0 5d Rep3

description: mRNA

source_name: seedlings

value: Seedlings

value: 5 days


title: CCl4+CHP_rep2

value: mouse liver


title: FACS-sorted cKIT(+) spermatogonia, Cbx2[23KRA/23KRA], H2AK119Ub1, rep3

description: CUT&RUN library

library_source: genomic

data_processing:
Paired-end fastq reads were quality trimmed (score 20), with stringency 1 using trim_galore (ver. 0.4.3). Trimmed reads were mapped to the mm10 genome using bowtie2 (ver. 2.3.1) with the end-to-end option.
Mapped reads were qualify-filtered with MAPQ higher than 10, and only properly-paired reads were retained using Samtools (ver. 1.4.1). Picard (ver. 2.17.1) was used to remove duplicate reads (http://broadinstitute.github.io/picard).
Alignment files (bam) were converted to bigwig files using Deeptools (ver. 2.2.4) by normalizing with RPKM and with binsize 10.
All analysis scripts are available at (github.com/jongminkmg/cbx2).
Assembly: mm10
Supplementary files format and content: bigwig
Library strategy: CUT&RUN

extract_protocol:
FACS purified, spermatogonia were attached to Poly-D-Lysine (R&D Systems, Cat# 3439-200-01) coated 24-well plates by 1 minute centrifugation at 80 × g (instead of binding to Concanavalin A beads). For H2AK119Ub1 Cut&Run, >100,000 c-Kit(+) spermatogonia were used per each replicate experiment. For CBX2 and RING1B Cut&Run, 20,000 THY1(+)&ITGa6(+) or c-Kit(+) spermatogonia were used for each replicate experiment. After the attachment, cells were fixed with 1% formaldehyde in PBS for 15 minutes. After two PBS washes and one PBST (0.05% Triton X-100 (v/v)) wash, cells were incubated with primary antibodies in PBST BSA (0.05% Triton X-100, 3% BSA) overnight at 4 ºC. Next day, cells were washed twice with PBST and finally washed with PBST EDTA (2 mM). Cells were then incubated with 500 ng/mL pA-MNase (protein A-MNase) in PBST BSA EDTA for more than 1 hour at 4 ºC. After pA-MNase binding, cells were washed with PBST EDTA twice, and the plate was placed on wet ice. Cells were then incubated in pre-chilled 200 µL PBST with 2 mM CaCl2 for 30 minutes. After MNase cutting, 200 µL stop solution (340 mM NaCl, 20 mM EDTA, 4 mM EGTA, 50 µg/µL RNase A) was added, and the plate was placed in a 37 ºC oven for 15 minutes. Released cut fragments were de-crosslinked in 0.1% SDS and 125 µg/mL Proteinase K (Sigma-Aldrich, Cat# 03115828001) in a 65 ºC oven for >12 hours. DNA was extracted using QIAquick PCR Purification Kit (Qiagen, Cat# 28104).
Illumina sequencing libraries were generated using NEBNext Ultra II DNA Library Prep Kit for Illumina kit (New England Biolabs, Cat# E7645) based on manufacturer's instructions. Adapters used in 0.6 µM working concentration. Final PCR cycles were between 9 (H2AK119Ub1, started from ~100,000 cells) to 11 (CBX2 and RING1B, started from ~20,000 cells). Number of PCR cycles were determined to be in the exponential phase by monitoring the reaction with SYBR green dye in a real time PCR machine.

treatment_protocol: No specific treatments

tag: cut&run antibody

value: H2AK119Ub1 (CST, clone D27C4, Cat# 8240)


title: CD11b+Ly6G+ S100A8CreCxcr2flox/flox + H. felis Mouse 1

type: RNA

city: Ann Arbor

first: Bioinformatics

last: Core

state: MI

address: BRCF Bioinformatics Core, 2800 Plymouth Rd

institute: University of Michigan

description: Gene expression data from gastric non-granulocytic-MDSC  after H. felis infection

hyb_protocol: Libraries were prepared using the NuGen WT-pico kit and hybridized on Affymetrix Mouse Gene ST 2.1

scan_protocol: GeneChips were scanned using the GeneChip Scanner 3000 7G

data_processing: Gene expression values were calculated using robust multi-array average (RMA)

source_name: 6mo H. felis-infected stomachs

label_protocol: Biotinylated cRNA were prepared according to the standard Affymetrix protocol from 6 ug total RNA (Expression Analysis Technical Manual, 2001, Affymetrix).

growth_protocol: After Helicobacter felis intection for 6 months, murine gastric strips from both the lesser and greater curvatures were prepared.

extract_protocol: RNA extracted using the RNEasy Microkit

treatment_protocol: Gastric cells were digested with Liberase, FACS was performed using FACSAria III and collected in PBS + 2% FBS.

value: granulocytic MDSC


title: CCL4_rep1

value: WT


title: CD11b+Ly6G+ Cxcr2flox/flox + H. felis Mouse 2

description: Gene expression data from gastric non-myeloid cells after H. felis infection


title: FACS-sorted cKIT(+) spermatogonia, Cbx2[23KRA/23KRA], H2AK119Ub1, rep2

description: CUT&RUN library

value: Testes


title: exp4_ch2_ADT_HTO

source_name: Channel 2; donors 4.1, 4.2, 4.3; unstim, TCR, cytokine, TCR+cytokine; 20h stimulation; ADT + HTO

value: Blood

value: MAIT cells


title: exp4_ch1_ADT_HTO

source_name: Channel 1; donors 4.1, 4.2, 4.3; unstim, TCR, cytokine, TCR+cytokine; 20h stimulation; ADT + HTO


title: CTRL_rep3

value: olive oil


title: CTRL_rep4


title: CD11b+Ly6G+ Cxcr2flox/flox + H. felis Mouse 1


title: CTRL_rep2


title: exp4_ch12_GEX

description: 10x Genomics

data_processing:
FASTQ files for all modalities (GEX, ADT/HTO, TCR) were processed using the Cell Ranger (v7.0.1) multi pipeline.
Assembly: GRCh38
Supplementary files format and content: Cell Ranger output files: barcodes.tsv.gz, features.tsv.gz, matrix.mtx.gz (GEX, ADT, HTO); filtered_contig_annotations.csv (TCR)
Supplementary files format and content: TotalSeq_C_Human_Universal_Cocktail_v1_137_Antibodies_399905_Barcodes.xlsx (TotalSeq-C Human Universal Cocktail antibodies and barcodes)

source_name: Channel 12; donors 4.1, 4.2, 4.3; unstim, TCR+cytokine; 68h stimulation; GEX

value: unstim, TCR+cytokine

value: 68 h stimulation

value: mRNA


title: exp4_ch11_GEX

description: 10x Genomics

source_name: Channel 11; donors 4.1, 4.2, 4.3; unstim, TCR+cytokine; 68h stimulation; GEX


title: CD11b-Ly6G- mouse 2

data_processing: Gene expression values were calculated using robust multi-array average (RMA)  (RMA) I

source_name: Cecum infected with  C. difficile strain VPI 10463 (ATCC 43255)

growth_protocol: After C. difficile intection for 2 days, murine ceca were biopsied.

treatment_protocol: Cells were digested with Liberase, FACS was performed using FACSAria III and collected in PBS + 2% FBS.

value: non-myeloid cells


title: CD11b+Ly6G+ S100A8CreCxcr2flox/flox + H. felis Mouse 2


title: exp4_ch10_GEX

description: 10x Genomics

source_name: Channel 10; donors 4.1, 4.2, 4.3; unstim, TCR+cytokine; 68h stimulation; GEX


title: CTRL_rep1

source_name: mouse liver


title: WD+CHP_rep5

data_processing:
After sequencing on the BGIseq-500 platform, the raw reads were filtered. Data filtering includes removing adaptor sequences, contamination and low-quality (phred quality < 20) reads from raw reads.
Sequenced reads were mapped to the GRCm38   mm10   primary   assembly  of the mouse genome using ensembl release 95 gene annotation with STAR aligner version 2.7.3a
Reads were counted using STAR aligner
Assembly: Mus_musculus.GRCm38.95
Supplementary files format and content: xlsx file with counts per gene per sample

value: liver

value: WD+CHP


title: CD11b+Ly6G+ mouse 1

value: granulocyte Myeloid-derived suppressor cells (MDSCs)


title: CD11b-Ly6G- mouse 1


title: exp4_ch3_ADT_HTO

source_name: Channel 3; donors 4.1, 4.2, 4.3; unstim, TCR, cytokine, TCR+cytokine; 20h stimulation; ADT + HTO

value: unstim, TCR, cytokine, TCR+cytokine

value: 20 h stimulation

value: ADT + HTO

value: TotalSeq-C0251 anti-human Hashtag 1 Antibody, TotalSeq-C0252 anti-human Hashtag 2 Antibody, TotalSeq-C0253 anti-human Hashtag 3 Antibody, TotalSeq-C0254 anti-human Hashtag 4 Antibody, TotalSeq-C0256 anti-human Hashtag 6 Antibody, TotalSeq-C0257 anti-human Hashtag 7 Antibody, TotalSeq-C0258 anti-human Hashtag 8 Antibody, TotalSeq-C0259 anti-human Hashtag 9 Antibody, TotalSeq-C0260 anti-human Hashtag 10 Antibody, TotalSeq-C0262 anti-human Hashtag 12 Antibody, TotalSeq-C0263 anti-human Hashtag 13 Antibody, TotalSeq-C0264 anti-human Hashtag 14 Antibody


title: FACS-sorted cKIT(+) spermatogonia, WT, H2AK119Ub1, rep3

description: CUT&RUN library

value: cKIT(+) differentiating spermatogonia

value: wild type

value: H2AK119Ub1 (CST, clone D27C4, Cat# 8240)


title: FACS-sorted cKIT(+) spermatogonia, WT, H2AK119Ub1, rep2

description: CUT&RUN library

value: Testes


title: FACS-sorted cKIT(+) spermatogonia, WT, RING1B, rep2a

description: CUT&RUN library

value: RING1B (CST, clone D22F2, Cat# 5694)


title: FACS-sorted cKIT(+) spermatogonia, WT, H2AK119Ub1, rep1

description: CUT&RUN library


title: FACS-sorted cKIT(+) spermatogonia, WT, RING1B, rep1a

description: CUT&RUN library


title: exp4_ch9_GEX

description: 10x Genomics

source_name: Channel 9; donors 4.1, 4.2, 4.3; unstim, TCR+cytokine; 68h stimulation; GEX

value: Blood

value: MAIT cells


title: exp4_ch8_GEX

description: 10x Genomics

source_name: Channel 8; donors 4.1, 4.2, 4.3; unstim, TCR, cytokine, TCR+cytokine; 20h stimulation; GEX


title: WD+CHP_rep3

value: WT


title: FACS-sorted cKIT(+) spermatogonia, Cbx2[23KRA/23KRA], H2AK119Ub1, rep1

description: CUT&RUN library


title: CD11b+Ly6G+ mouse 2


title: CD11b-Ly6G+ mouse 1

description: Gene expression data from gastric non-myeloid control  after H. felis infection

value: non-myeloid control


title: exp4_ch7_GEX

description: 10x Genomics

source_name: Channel 7; donors 4.1, 4.2, 4.3; unstim, TCR, cytokine, TCR+cytokine; 20h stimulation; GEX

value: mRNA


title: CD11b+Ly6G+ mouse 2

description: Gene expression data from gastric granulocytic-MDSC  after H. felis infection

value: granulocytic MDSC


title: WD+CHP_rep2

value: liver

value: WD+CHP


title: FACS-sorted cKIT(+) spermatogonia, WT, CBX2-856, rep1a

description: CUT&RUN library

value: CBX2 (home-made, 856)


title: CD11b+Ly6G- mouse 2

value: non-granulocytic-MDSC


title: exp4_ch6_GEX

description: 10x Genomics

source_name: Channel 6; donors 4.1, 4.2, 4.3; unstim, TCR, cytokine, TCR+cytokine; 20h stimulation; GEX

value: unstim, TCR, cytokine, TCR+cytokine

value: 20 h stimulation


title: FACS-sorted cKIT(+) spermatogonia, WT, CBX2-856, rep2a

description: CUT&RUN library

value: cKIT(+) differentiating spermatogonia


title: CD11b+Ly6G+ mouse 1


title: WD+water_rep5

value: WD+water


title: exp4_ch5_GEX

description: 10x Genomics

source_name: Channel 5; donors 4.1, 4.2, 4.3; unstim, TCR, cytokine, TCR+cytokine; 20h stimulation; GEX


title: WD+water_rep4


title: exp4_ch4_GEX

description: 10x Genomics

source_name: Channel 4; donors 4.1, 4.2, 4.3; unstim, TCR, cytokine, TCR+cytokine; 20h stimulation; GEX


title: WD+CHP_rep4


title: FACS-sorted THY1(+)&ITGa6(+) spermatogonia, WT, RING1B, rep1

description: CUT&RUN library

value: wild type


title: FACS-sorted THY1(+)&ITGa6(+) spermatogonia, WT, RING1B, rep2

description: CUT&RUN library

value: Testes


title: xMG-8weeks, rep3

city: La Jolla

first: Christopher

last: Glass

state: CA

address: 9500 Gilman Dr.

department: CMM

institute: University of California, San Diego

description:
iPSCModels_raw.txt
iPSCModels_TPM.txt

data_processing:
RNA-seq data was mapped to hg38/mm10 genome using STAR (Dobin et al., 2013) with default parameters. HOMER’s analyzeRepeats was used to calculate gene expression raw counts with the option “-condenseGenes -count exons -noadj” and transcript per kilobase million (TPM) with the option “-count exons -tpm”. Genes shorter than 200bp were removed. TPMs were matched to raw counts using accession numbers.
Assembly: hg38
Supplementary files format and content: iPSCModels_raw.txt, tab-delimited text file containing table of unnormalized read counts ffor all RNA-seq samples
Supplementary files format and content: iPSCModels_TPM.txt, tab-delimited text file containing table of TPM counts for all RNA-seq samples

extract_protocol:
For iMGs, cells were carefully manually scraped from the Matrigel coated plate and were concentrated by a 300 rcf x 5 minutes. For oMGs, organoids were carefully collected, allowed to settle by gravity, and then dissociated mechanically in staining buffer (HBSS 1x with 1mM EDTA and 1% BSA) using a 2 ml polytetrafluoroethylene pestle. iMGs and oMGs were stained with the following Biolegend antibodies/dyes: CD64-APC (305014), CX3CR1 PCP-Cy5.5 (341614), CD14-488 (325610), CD11b-PE (301309), HLADR PE-Cy7 (307616), CD45-APC-CY7 (368516), and Zombie Violet (1:1000, Biolegend) for live/dead discrimination. xMGs were isolated and sorted at indicated time points following xenotransplantation. Briefly, mouse brains were homogenized as previously described by gentle mechanical dissociation (Gosselin et al., 2014) and stained with DAPI, CX3CR1-APC (Biolegend 149008), anti-human CD11b-PE (Biolegend 301306), anti-human CD45-FITC (Biolegend 368508). Microglia were purified with either a BD Influx  or FACS  AriaFusion.TA
RNA-seq libraries were prepared as previously described (Gosselin et al., 2017). Briefly, mRNAs were incubated with Oligo d(T) Magnetic Beads (New England BioLabs S1419), then fragmented in 2x Superscript III first-strand buffer (Thermo Fisher Scientific with 10mM DTT (Thermo Fisher Scientific 18080044) at 94oC for 9 minutes. Fragment mRNA was then incubated with 0.5 μl of Random primers (3 mg/mL) (Thermo Fisher Scientific 48190011), 0.5 μl of 50mM Oligo dT primer, (Thermo Fisher Scientific, 18418020), 0.5 μl of SUPERase-In (Thermo Fisher Scientific AM2694), 1 μl of dNTPs (10 mM) at 50°C for one minute. Then, 1 μl of 10mM DTT, 6 μl of H2O+0.02%Tween-20 (Sigma), 0.1 μl Actinomycin D (2 mg/mL), and 0.5 μl of Superscript III (Thermo Fisher Scientific) were added to the mixture. cDNA was then generated by incubating mixture in a PCR machine at the following conditions: 25oC for 10 minutes, 50°C for 50 minutes, and a 4°C hold. Product was purified using RNAClean XP beads (Beckman Coulter A63987) according to manufacturer’s instructions and eluted with 10 μl of nuclease-free H2O. Eluate was then incubated with 1.5 μl of Blue Buffer (Enzymatics P7050L), 1.1 μl of dUTP mix (10 mM dATP, dCTP, dGTP and 20 mM dUTP), 0.2 mL of RNase H (5 U/mL Y9220L), 1.2 μl of H2O+0.02%Tween-20, and 1 μl of DNA polymerase I (Enzymatics P7050L) at 16°C overnight. DNA was then purified using 3 μl of SpeedBeads (Thermo Scientific Fisher 651520505025) resuspended in 28 μl of 20% PEG8000/2.5M NaCl to final of 13% PEG. DNA eluted with 40 mL nuclease free H2O+0.02%Tween-20 and underwent end repair by blunting, A-tailing and adaptor ligation (Heinz et al., 2010a) as previously described using barcoded adapters. Libraries were PCR-amplified for 12-15 cycles, size selected by gel extraction for 200-500 bp, and sequenced on a HiSeq 4000 (Illumina) or a NextSeq 500 (Illumina) for 51 cycles.

value: Xenotransplanted iPSC-microglia

value: 8weeks post-xenotransplantation


title: exp4_ch2_GEX

description: 10x Genomics

source_name: Channel 2; donors 4.1, 4.2, 4.3; unstim, TCR, cytokine, TCR+cytokine; 20h stimulation; GEX

value: Blood

value: MAIT cells


title: exp4_ch3_GEX

description: 10x Genomics

source_name: Channel 3; donors 4.1, 4.2, 4.3; unstim, TCR, cytokine, TCR+cytokine; 20h stimulation; GEX


title: CD11b+Ly6G- mouse 1

source_name: 6mo H. felis-infected stomachs


title: FACS-sorted THY1(+)&ITGa6(+) spermatogonia, WT, CBX2-856, rep1

description: CUT&RUN library


title: WD+water_rep2

value: WT


title: xMG-8weeks, rep2

description:
iPSCModels_raw.txt
iPSCModels_TPM.txt


title: WD+water_rep3


title: FACS-sorted cKIT(+) spermatogonia, Rosa26-CreERT2; Cbx2[Flox/+], +Mock, rep2

data_processing:
For Cbx2KO experiments, low quality reads (Phred score 20) and adapter containing reads (stringency 1) were trimmed using trim_galore (ver. 0.4.3) (Martin 2011). Trimmed reads were mapped to the mouse genome (mm10) with default parameters using STAR (ver. 2.5.3) (Dobin et al. 2013). Mapped reads were filtered with quality score 10 (-q 10), and only properly paired reads were retained (-f 2) using Samtools (ver. 1.4.1) (Li et al. 2009). Counts per gene was obtained using featureCounts function of the subread package (ver. 1.5.0) (Liao et al. 2014). Data from Cbx223KRA experiments were processed identically as the Cbx2KO experiment, except counts per gene were obtained with the strand parameter ‘-s 2’ of featureCounts to account for the stranded library.
Assembly: mm10
Supplementary files format and content: Tab-separated counts files

source_name: Testes

extract_protocol:
extract protocol
We used SMART-Seq mRNA LP (Takara, Cat# 634768) following the manufacturer’s manual to make the RNA-seq libraries for the Cbx2[iKO] experiments. Cells from two control (R26-CreERT2; Cbx2Flox/+, mock injected) and two experimental (R26-CreERT2/+; Cbx2Δ/Flox, tamoxifen injected) animals were used. Equal amounts of total RNA (20 ng) was used as the starting material for each library. cDNA was amplified with 7 PCR cycles. Equal amounts of cDNA (1.3 ng) were used for library preparation. A total of 11 cycles of library amplifying, index PCRs were performed.

value: Rosa26-CreERT2; Cbx2[Flox/+]

value: 200uL corn oil (Mock) 3X IP injections


title: exp4_ch1_GEX

description: 10x Genomics

source_name: Channel 1; donors 4.1, 4.2, 4.3; unstim, TCR, cytokine, TCR+cytokine; 20h stimulation; GEX

value: mRNA


title: CD11b-Ly6G- mouse 2

value: non-myeloid cells


title: CD11b-Ly6G- mouse 1


title: WD+CHP_rep1

value: liver

value: WD+CHP


title: WD+water_rep1


title: exp2_ch8_TCR

description: 10x Genomics

extract_protocol:
MAIT (CD3+MR1/5-OP-RU+) and Tmem (CD3+MR1/5-OP-RU-CCR7-) cells were sorted from human blood (n = 8) and liver (n = 3) samples for single-cell gene, surface protein and TCR repertoire analysis using 10x Genomics technology.
Sequencing libraries were generated using the 10x Genomics Chromium Next GEM Single Cell 5' Reagent Kits v2 (Dual Index).

value: Blood and liver

tag: samples

value: Blood 2.2, Liver 2.2, Blood 2.4, Blood 2.6, Blood 2.7

value: MAIT and Tmem cells

value: TCR


title: FACS-sorted cKIT(+) spermatogonia, Rosa26-CreERT2; Cbx2[Flox/Δ], +TAM, rep2

extract_protocol:
More than 100,000 c-KIT(+) cells were isolated, centrifuged to remove sorting buffer. Cell pellets were snap frozen in liquid nitrogen and stored in -80 ºC. RNA was isolated using Trizol-based phase separation, followed by column-based purification (Qiagen RNeasy mini, Cat# 74104).
We used SMART-Seq mRNA LP (Takara, Cat# 634768) following the manufacturer’s manual to make the RNA-seq libraries for the Cbx2[iKO] experiments. Cells from two control (R26-CreERT2; Cbx2Flox/+, mock injected) and two experimental (R26-CreERT2/+; Cbx2Δ/Flox, tamoxifen injected) animals were used. Equal amounts of total RNA (20 ng) was used as the starting material for each library. cDNA was amplified with 7 PCR cycles. Equal amounts of cDNA (1.3 ng) were used for library preparation. A total of 11 cycles of library amplifying, index PCRs were performed.

tag: tissue

value: cKIT(+) differentiating spermatogonia

value: Rosa26-CreERT2; Cbx2[Flox/{delta}]

value: 200uL 20mg/mL Tamoxifen 3X IP injections


title: xMG-8weeks, rep1

description:
iPSCModels_raw.txt
iPSCModels_TPM.txt

tag: cell type

value: Xenotransplanted iPSC-microglia

value: 8weeks post-xenotransplantation


title: CD+water_rep5

value: CD+water


title: FACS-sorted cKIT(+) spermatogonia, Rosa26-CreERT2; Cbx2[Flox/Δ], +TAM, rep1

value: Testes

tag: strain


title: xMG-3weeks, rep3

description:
iPSCModels_raw.txt
iPSCModels_TPM.txt

value: 3weeks post-xenotransplantation


title: FACS-sorted cKIT(+) spermatogonia, Rosa26-CreERT2; Cbx2[Flox/+], +Mock, rep1

tag: genotype

value: Rosa26-CreERT2; Cbx2[Flox/+]

value: 200uL corn oil (Mock) 3X IP injections


title: xMG-3weeks, rep1

description:
iPSCModels_raw.txt
iPSCModels_TPM.txt

tag: treatment


title: exp2_ch7_TCR

description: 10x Genomics


title: CD+water_rep3

value: WT


title: exp2_ch6_TCR

description: 10x Genomics


title: xMG-12days, rep4

description:
iPSCModels_raw.txt
iPSCModels_TPM.txt

value: 12days post-xenotransplantation


title: CD+water_rep4

value: liver


title: CD+water_rep1


title: CD+water_rep2


title: FACS-sorted THY1(+)&ITGa6(+) spermatogonia, WT, CBX2-856, rep2

description: CUT&RUN library

value: wild type

value: CBX2 (home-made, 856)


title: exp2_ch3_TCR

description: 10x Genomics

value: Blood and liver

value: Blood 2.1, Liver 2.1, Blood 2.3, Liver 2.3, Blood 2.5, Blood 2.8

value: MAIT and Tmem cells


title: xMG-12days, rep2

description:
iPSCModels_raw.txt
iPSCModels_TPM.txt


title: FACS-sorted cKIT(+) spermatogonia, Cbx2 amplicon, Mut band, Rosa26-CreERT2; Cbx2[Flox/Flox], rep1

description:
Cbx2 amplicon generated using cell-barcoded cDNA
Cbx2_iKO_amplicon_MutBand_rep1

data_processing:
1) Wild-type adult testis profiling: Cellranger count (cellranger/2.0.0) was used to map fastq files to the mouse genome (refdata-cellranger=mm10-1.2.0).
2) Cbx2 inducible knock out & FACS-sorted cKIT(+) spermatogonia profiling : Cellranger count (cellranger/6.0) was used to map fastq files to the mouse genome (refdata-cellranger-mm10-3.0.0) and to obtain gene expression counts per cell. Cells were filtered by a) nFeature_RNA > 2000, b) mitochondrial RNA percentage < 7.5, c) percent occupied by the largest gene < 3, and d) RNA count and feature ratio < 5.5 (see scripts posted on https://github.com/jongminkmg/Cbx2). Two control and two inducible KO samples were integrated to align cell populations across all four samples. Integrated data were then scaled and processed with 30 principal components for clustering. Differential gene expression tests were performed by the FindMarkers function of Seurat using Wilcoxon rank sum test.
Cbx2 genotypes were assigned based on independent Cbx2 amplicon sequencing data using a custom Python script (https://github.com/jongminkmg/Cbx2).  The script identifies 'TGGCGCATCTGGTTCCTTGAGCTTGGAGCG' (WT, exon5 linked to exon4) or 'TGGCGCATCTGGTTCTTGGAGGACCAGCCG' (Mut, exon5 linked to exon2) from the read2 of the paired-end reads, and assigns the genotypes to the associated read1 (UMI-barcode). When an UMI is assigned to two different genotypes, likely due to PCR chimera formation, the genotype was left unassigned unless reads supporting one genotype are present 10 times more than the other genotype. A cell was assigned to be 'Mut' when all Cbx2 amplicon reads show recombination (exon5 linked to exon2), and otherwise assigned to be 'WTorHet'. Only the cells that have at least four UMIs linked to Cbx2 sequence were assigned genotypes. The metadata for their genotypes (WT/Het or Mut) were added to the Seurat object by the AddMetaData function.
Supplementary files format and content: Tab-separated values files and matrix files

extract_protocol:
1) Wild-type adult testis profiling: dissociated cells were diluted to 1000 cells/µL, and total 5200 cells (5.2 µL) were loaded into the Chromium controller to obtain RNA-seq profiles of approximately 3000 cells.   2) Cbx2 inducible knock out & FACS-sorted cKIT(+) spermatogonia profiling : c-KIT(+) spermatogonia were FACS-sorted to concentration of  about 300~600 cells/µL, and a total ~8000 cells were loaded into the Chromium controller. We used 5' barcoding instead of 3' barcoding because deleted exons of Cbx2 were close to the 5' end of the gene. Single cell analysis with targeted genotyping was done by adapting 'Genotyping of Transcriptome (GoT)' (Nam et al., 2019, PMID: 31270458) and V(D)J region amplification of T or B-cell receptor protocol by the 10X Genomics. We aimed to make specific Cbx2 amplicons from cell-barcoded cDNA to amplify cDNA fragments and sequence Exon5 junctions to distinguish wild type (Exon4 spliced to Exon5) and mutant (Exon2 spliced to Exon5 after the deletion of Exon3, 4) for specific cell barcodes.
1) Wild-type adult testis profiling: we used Chromium Single Cell 3’ Library & Gel Bead Kit v2 (10X Genomics, Cat: 120267) for single cell RNA-seq from wild-type adult testes. After producing emulsions encapsulating gel beads and cells in the Chromium controller, reverse transcription and 12 cycles of PCR were performed to produce cell-barcoded amplified cDNA. Illumina sequencing-ready libraries were produced from 200 ng of the amplified cDNA based on the manufacturer’s protocol. 12 cycles of library amplification indexing PCR was performed.   2) Cbx2 inducible knock out & FACS-sorted cKIT(+) spermatogonia profiling : single cell barcoded cDNA was made with Chromium Next GEM Single Cell 5' Reagent Kits (10X Genomics, Cat#:1000265) based on the manufacturer's protocol with 14 PCR cycles of cDNA amplification. 50 ng of the amplified cDNA based on the manufacturer’s protocol. 14 cycles of library amplification indexing PCR was performed. To generate Cbx2 amplicons, we performed nested PCRs to amplify Cbx2-specific cDNAs with cell barcodes. In the second PCR step of the nested PCR, Cbx2-specific primer that has a partial Illumina read2 sequence was used. The Cbx2-specific second nested primer also contains 'stagger' sequences between the Cbx2-specific region and partial read2 to increase library complexity for accurate base calls during Illumina sequencing. We confirmed the amplification of the correct-sized bands corresponding to wild-type and deleted Cbx2 amplicons by agarose gel electrophoresis. The amplified bands were excised and gel purified. Index PCR was performed to make the final Illumina-compatible libraries with unique indices with 10X provided index-PCR primers. Cbx2 amplicon libraries were pooled with standard 10X single cell gene expression libraries to occupy ~2% of total reads. A total of 4 samples were sequenced in one P3 lane on a NextSeq 2000 (NextSeq 2000 P3 Reagents (100 Cycles) Cat:20040559).

value: cKIT(+) differentiating spermatogonia

value: Rosa26-CreERT2; Cbx2[Flox/Flox]

value: 150uL 20mg/mL Tamoxifen 3X IP injections


title: FACS-sorted cKIT(+) spermatogonia, Cbx2 amplicon, WT band, Rosa26-CreERT2; Cbx2[Flox/Flox], rep2

description:
Cbx2 amplicon generated using cell-barcoded cDNA
Cbx2_iKO_amplicon_WTBand_rep2


title: xMG-12days, rep3

description:
iPSCModels_raw.txt
iPSCModels_TPM.txt

value: Xenotransplanted iPSC-microglia


title: exp2_ch2_TCR

description: 10x Genomics

value: TCR


title: FACS-sorted cKIT(+) spermatogonia, Cbx2 amplicon, WT band, Rosa26-CreERT2; Cbx2[Flox/Flox], rep1

description:
Cbx2 amplicon generated using cell-barcoded cDNA
Cbx2_iKO_amplicon_WTBand_rep1

value: Testes


title: xMG-3weeks, rep2

description:
iPSCModels_raw.txt
iPSCModels_TPM.txt

value: 3weeks post-xenotransplantation


title: FACS-sorted cKIT(+) spermatogonia, Cbx2[23KRA/23KRA], H3K27me3, rep1

description:
CUT&RUN library
CBX2KRA_H3K27me3_rep1

data_processing:
Paired-end fastq reads were quality trimmed (score 20), with stringency 1 using trim_galore (ver. 0.4.3). Trimmed reads were mapped to the mm10 genome using bowtie2 (ver. 2.3.1) with the end-to-end option.
Mapped reads were qualify-filtered with MAPQ higher than 10, and only properly-paired reads were retained using Samtools (ver. 1.4.1). Picard (ver. 2.17.1) was used to remove duplicate reads (http://broadinstitute.github.io/picard).
Alignment files (bam) were converted to bigwig files using Deeptools (ver. 2.2.4) by normalizing with RPKM and with binsize 10.
Peaks were identified by using MACS2 (ver. 2. 1) with the ‘--broad' option, by comparing CUT&RUN results using CBX2 antibodies (Experimental) to IgG (Control) or using HA antibodies applied to Cbx2HA/HA (Experimental) or Cbx2+/+ (Control) mice.
All analysis scripts are available at (github.com/jongminkmg/cbx2).
Assembly: mm10
Supplementary files format and content: bigwig
Library strategy: CUT&RUN-seq

extract_protocol:
FACS purified, >100,000 c-Kit(+) spermatogonia were attached to Poly-D-Lysine (R&D Systems, Cat# 3439-200-01) coated 24-well plates by 1 minute centrifugation at 80 × g (instead of binding to Concanavalin A beads). After the attachment, cells were fixed with 1% formaldehyde in PBS for 15 minutes. After two PBS washes and one PBST (0.05% Triton X-100 (v/v)) wash, cells were incubated with primary antibodies in PBST BSA (0.05% Triton X-100, 3% BSA) overnight at 4 ºC. Next day, cells were washed twice with PBST and finally washed with PBST EDTA (2 mM). Cells were then incubated with 500 ng/mL pA-MNase (protein A-MNase) in PBST BSA EDTA for more than 1 hour at 4 ºC. After pA-MNase binding, cells were washed with PBST EDTA twice, and the plate was placed on wet ice. Cells were then incubated in pre-chilled 200 µL PBST with 2 mM CaCl2 for 30 minutes. After MNase cutting, 200 µL stop solution (340 mM NaCl, 20 mM EDTA, 4 mM EGTA, 50 µg/µL RNase A) was added, and the plate was placed in a 37 ºC oven for 15 minutes. Released cut fragments were de-crosslinked in 0.1% SDS and 125 µg/mL Proteinase K (Sigma-Aldrich, Cat# 03115828001) in a 65 ºC oven for >12 hours. DNA was extracted using phenol-chloroform phase separation and sodium acetate/ethanol precipitation. Resuspended DNA was further cleaned up by using 2.0X Solid Phase Reversible Immobilization (SPRI) beads to remove residual salts.
Illumina sequencing libraries were generated based on a published protocol (Bowman et al., 2013) with the following modifications. SPRI beads were used in 2.0X ratio following end-repair and A-tailings steps instead of 1.8X. Adapters used in 2 nM final concentration instead of 10 nM. PCR extension was done for 30 seconds instead of 45 seconds. Final PCR cycles were between 12 to 17. Number of PCR cycles were determined to be in the exponential phase by a pre-run of 10% of reaction with SYBR green dye in a real time PCR machine.

value: H3K27me3 (CST, clone C36B11, Cat# 9733)


title: FACS-sorted cKIT(+) spermatogonia, Cbx2 amplicon, Mut band, Rosa26-CreERT2; Cbx2[Flox/Flox], rep2

description:
Cbx2 amplicon generated using cell-barcoded cDNA
Cbx2_iKO_amplicon_MutBand_rep2


title: exp2_ch5_TCR

description: 10x Genomics

value: Blood 2.2, Liver 2.2, Blood 2.4, Blood 2.6, Blood 2.7


title: exp2_ch4_TCR

description: 10x Genomics


title: FACS-sorted cKIT(+) spermatogonia, Cbx2[Flox/Flox], rep1

description:
Used Chromium Next GEM Single Cell 5' Reagent Kit by 10X Genomics
Cbx2_WT_rep1


title: oMG, rep6

description:
iPSCModels_raw.txt
iPSCModels_TPM.txt

value: iPSC-microglia co-cultured with cerebral organoid


title: FACS-sorted cKIT(+) spermatogonia, Cbx2[Flox/Flox], rep2

description:
Used Chromium Next GEM Single Cell 5' Reagent Kit by 10X Genomics
Cbx2_WT_rep2


title: FACS-sorted cKIT(+) spermatogonia, Rosa26-CreERT2; Cbx2[Flox/Flox], rep2

description:
Used Chromium Next GEM Single Cell 5' Reagent Kit by 10X Genomics
Cbx2_iKO_rep2


title: exp2_ch5_ADT_HTO

description:
10x Genomics
Processed data included on GEX samples

source_name: Channel 5; donors 2.2, 2.4, 2.6, 2.7; ADT + HTO

value: ADT + HTO

value: TotalSeq-C0257 anti-human Hashtag 7 Antibody, TotalSeq-C0259 anti-human Hashtag 9 Antibody, TotalSeq-C0260 anti-human Hashtag 10 Antibody, TotalSeq-C0262 anti-human Hashtag 12 Antibody, TotalSeq-C0263 anti-human Hashtag 13 Antibody


title: exp2_ch4_ADT_HTO

source_name: Channel 4; donors 2.1, 2.3, 2.5, 2.8; ADT + HTO

value: TotalSeq-C0251 anti-human Hashtag 1 Antibody, TotalSeq-C0252 anti-human Hashtag 2 Antibody, TotalSeq-C0253 anti-human Hashtag 3 Antibody, TotalSeq-C0254 anti-human Hashtag 4 Antibody, TotalSeq-C0255 anti-human Hashtag 5 Antibody, TotalSeq-C0256 anti-human Hashtag 6 Antibody


title: xMG-12days, rep1

description:
iPSCModels_raw.txt
iPSCModels_TPM.txt

value: 12days post-xenotransplantation


title: Psoriasis-lesional skin, human [LE-18-TI-28-PSO]

city: Leipzig

first: Maria

last: Schmidt

address: Härtelstr. 16-18

country: Germany

institute: IZBI

data_processing:
Demultiplexing was done using bcl2fastq (Illumina®, v2.20.0.422).
For further analysis and read alignment CellRanger software v7.1.0 was used with default settings as suggested by 10x Genomics.
Assembly: GRCh38-2020-A
Supplementary files format and content: Filtered feature-barcode matrices in Market Exchange Format (MEX) outputted by cellranger. Each element of the feature-barcode matrix is the number of UMIs associated with a feature (row) and a cell-associated barcode (column).
Supplementary files format and content: TSV files with feature and barcode sequences corresponding to row and column indices, respectively, outputted by cellranger.

source_name: Psoriasis-lesional skin, human

extract_protocol:
Cell suspensions were generated using the Whole Skin Dissociation Kit (Miltenyi®, Bergisch Gladbach, Germany) according to the manufacturer’s specifications. Single-cell suspensions were subsequently counted for the number of dead cells by Trypan blue staining (Sigma Aldrich, Munich) which did not exceed 5% of cells.
Single cells were loaded on a 10x Genomics®) Chromium Next GEM Chip G (10x Genomics®, Pleasanton, CA, U.S.A.) according to the manufacturer’s specifications. Overall, 10,000 cells were loaded into one channel of the Chromium system using the v3.1 single-cell reagent kits (10x Genomics®). Following capture and lysis, cDNA was synthesized and amplified for 12 PCR cycles. The amplified cDNA was then used to construct Illumina®) sequencing libraries according to the instruction of the Chromium User Guide® (10x Genomics®). Briefly, blunt end repair was followed by adapter ligation. During PCR amplification, sample indices were added. The barcoded libraries were purified with SPRI select beads and quantified using Qubit®) dsDNA HS Assay Kit (Invitrogen, Thermo Fisher Scientific, Waltham, Massachusetts, USA) according to the manufacturer’s instructions. Correct size distribution of the library DNA was analyzed using the Fragment Analyzer (Agilent®), Santa Clara, CA, U.S.A.).
Sequencing of 2x150 bp was performed with an Illumina®) NextSeq 550 sequencer (Illumina®), San Diego, CA, USA). Demultiplexing was done using bcl2fastq v2.20.0.422 (Illumina®).

value: Skin

value: male

tag: age

tag: pasi

tag: pretreatment

value: No pretreatment


title: FACS-sorted cKIT(+) spermatogonia, Rosa26-CreERT2; Cbx2[Flox/Flox], rep1

description:
Used Chromium Next GEM Single Cell 5' Reagent Kit by 10X Genomics
Cbx2_iKO_rep1

value: cKIT(+) differentiating spermatogonia


title: exp2_ch1_TCR

description: 10x Genomics

value: Blood and liver

value: Blood 2.1, Liver 2.1, Blood 2.3, Liver 2.3, Blood 2.5, Blood 2.8

value: MAIT and Tmem cells


title: exp2_ch6_ADT_HTO

source_name: Channel 6; donors 2.2, 2.4, 2.6, 2.7; ADT + HTO


title: xMG-7days, rep1

description:
iPSCModels_raw.txt
iPSCModels_TPM.txt

tag: cell line

value: Xenotransplanted iPSC-microglia

value: 7days post-xenotransplantation


title: Psoriasis-lesional skin, human [LE-15-TI-19-PSO]

tag: Sex

value: female


title: FACS-sorted cKIT(+) spermatogonia, WT, CBX2-856, rep1

description:
CUT&RUN library
WT_CBX2-856_rep1

value: Testes

value: wild type

value: CBX2 (home-made, 856)


title: exp2_ch7_ADT_HTO

source_name: Channel 7; donors 2.2, 2.4, 2.6, 2.7; ADT + HTO

value: Blood 2.2, Liver 2.2, Blood 2.4, Blood 2.6, Blood 2.7

value: ADT + HTO


title: FACS-sorted cKIT(+) spermatogonia, WT, H3K4me3, rep1

description:
CUT&RUN library
WT_H3K4me3_rep1

value: H3K4me3 (Millipore, Cat# 07-473)


title: oMG, rep4

description:
iPSCModels_raw.txt
iPSCModels_TPM.txt

value: iPSC-microglia co-cultured with cerebral organoid


title: oMG, rep5

description:
iPSCModels_raw.txt
iPSCModels_TPM.txt


title: FACS-sorted cKIT(+) spermatogonia, Cbx2[23KRA/23KRA], CBX2-856, rep1

description:
CUT&RUN library
CBX2KRA_CBX2-856_rep1


title: FACS-sorted cKIT(+) spermatogonia, WT, PHC2, rep2

description:
CUT&RUN library
WT_PHC2_rep2

value: PHC2 (CST, H8523-ne-1.8, discontinued)


title: xMG-7days, rep3

description:
iPSCModels_raw.txt
iPSCModels_TPM.txt


title: exp2_ch8_ADT_HTO

source_name: Channel 8; donors 2.2, 2.4, 2.6, 2.7; ADT + HTO

value: TotalSeq-C0257 anti-human Hashtag 7 Antibody, TotalSeq-C0259 anti-human Hashtag 9 Antibody, TotalSeq-C0260 anti-human Hashtag 10 Antibody, TotalSeq-C0262 anti-human Hashtag 12 Antibody, TotalSeq-C0263 anti-human Hashtag 13 Antibody


title: Psoriasis-lesional skin, human [LE-13-TI-10-PSO]

value: Skin

value: Methotrexate 15 mg weekly


title: FACS-sorted cKIT(+) spermatogonia, WT, H3K27me3, rep1

description:
CUT&RUN library
WT_H3K27me3_rep1

value: cKIT(+) differentiating spermatogonia

value: H3K27me3 (CST, clone C36B11, Cat# 9733)


title: FACS-sorted cKIT(+) spermatogonia, WT, H3K4me3, rep2

description:
CUT&RUN library
WT_H3K4me3_rep2


title: xMG-7days, rep2

description:
iPSCModels_raw.txt
iPSCModels_TPM.txt


title: oMG, rep3

description:
iPSCModels_raw.txt
iPSCModels_TPM.txt


title: exp2_ch3_ADT_HTO

source_name: Channel 3; donors 2.1, 2.3, 2.5, 2.8; ADT + HTO

value: Blood and liver

value: Blood 2.1, Liver 2.1, Blood 2.3, Liver 2.3, Blood 2.5, Blood 2.8

value: MAIT and Tmem cells

value: TotalSeq-C0251 anti-human Hashtag 1 Antibody, TotalSeq-C0252 anti-human Hashtag 2 Antibody, TotalSeq-C0253 anti-human Hashtag 3 Antibody, TotalSeq-C0254 anti-human Hashtag 4 Antibody, TotalSeq-C0255 anti-human Hashtag 5 Antibody, TotalSeq-C0256 anti-human Hashtag 6 Antibody


title: Psoriasis-lesional skin, human [LE-12-TI-06-PSO]

value: male

value: No pretreatment


title: exp2_ch2_ADT_HTO

source_name: Channel 2; donors 2.1, 2.3, 2.5, 2.8; ADT + HTO


title: FACS-sorted cKIT(+) spermatogonia, WT, PHC2, rep1

description:
CUT&RUN library
WT_PHC2_rep1

value: Testes

value: wild type


title: Psoriasis-lesional skin, human [LE-16-TI-02-PSO]


title: FACS-sorted cKIT(+) spermatogonia, WT, BMI1, rep2

description:
CUT&RUN library
WT_BMI1_rep2

value: BMI1 (Bethyl lab., Cat# A301-694A)


title: oMG, rep2

description:
iPSCModels_raw.txt
iPSCModels_TPM.txt


title: oMG, rep1

description:
iPSCModels_raw.txt
iPSCModels_TPM.txt


title: exp2_ch1_ADT_HTO

source_name: Channel 1; donors 2.1, 2.3, 2.5, 2.8; ADT + HTO

value: ADT + HTO


title: iMG, rep6

description:
iPSCModels_raw.txt
iPSCModels_TPM.txt

value: iPSC-microglia


title: FACS-sorted cKIT(+) spermatogonia, WT, BMI1, rep1

description:
CUT&RUN library
WT_BMI1_rep1


title: exp2_ch7_GEX

description: 10x Genomics

value: Blood 2.2, Liver 2.2, Blood 2.4, Blood 2.6, Blood 2.7

value: mRNA


title: BF_LE_03_VC_03_all

data_processing:
Demultiplexing was done using bcl2fastq (Illumina®, v2.20.0.422).
For further analysis and read alignment CellRanger software v3.1.0 was used with default settings as suggested by 10x Genomics.
Assembly: GRCh38-3.0.0
Supplementary files format and content: Filtered feature-barcode matrices in Market Exchange Format (MEX) outputted by cellranger. Each element of the feature-barcode matrix is the number of UMIs associated with a feature (row) and a cell-associated barcode (column).
Supplementary files format and content: TSV files with feature and barcode sequences corresponding to row and column indices, respectively, outputted by cellranger.

value: Skin

value: topical therapy with glucocorticoid


title: FACS-sorted cKIT(+) spermatogonia, WT, RING1B, rep2

description:
CUT&RUN library
WT_RING1B_rep2

value: cKIT(+) differentiating spermatogonia

value: RING1B (CST, clone D22F2, Cat# 5694)


title: exp2_ch8_GEX

description: 10x Genomics

value: Blood and liver


title: BF-LE-01-KT-PSO_all

value: untreated


title: iMG, rep5

description:
iPSCModels_raw.txt
iPSCModels_TPM.txt


title: exp2_ch6_GEX

description: 10x Genomics

value: MAIT and Tmem cells


title: FACS-sorted cKIT(+) spermatogonia, Cbx2[HA/HA], HA-16B12

description:
CUT&RUN library
HACBX2_HA-16B12

value: HA (Biolegend Inc, clone 16B12, Cat# 901502)


title: iMG, rep4

description:
iPSCModels_raw.txt
iPSCModels_TPM.txt


title: FACS-sorted cKIT(+) spermatogonia, WT, RING1B, rep1

description:
CUT&RUN library
WT_RING1B_rep1


title: iMG, rep3

description:
iPSCModels_raw.txt
iPSCModels_TPM.txt


title: R27

city: Rensselaer

first: Farhad

last: Farjood

address: 1 Discovery Drive

institute: Neural Stem Cell Institute

data_processing:
Bam files were then read into R using the GenomicAlignments package to generate a counts matrix for further analysis
Assembly: hg19
Supplementary files format and content: Rdata file with count matrices for each sample

source_name: RPE

growth_protocol: Cultures were maintained in RPE medium: MEM-a modified medium (Sigma-Aldrich), 2 mM L-glutamine, penicillin/streptomycin (1:100), 1% Na-Pyruvate, 10% fetal bovine serum supplemented with THT (Taurine, Hydrocortisone, Triiodo-thyronin), and N1 medium supplement (Sigma-Aldrich). Cells were incubated in a humidified incubator at 37oC and 5% CO2 and the medium was replaced every 3 days

extract_protocol:
RNA was harvested using Direct-zol RNA kit (Zymo Research)
Library preparation was then carried out with the TruSeq Stranded Total RNA kit (Illumina)

value: RPE

value: WT

value: 8 weeks


title: FACS-sorted cKIT(+) spermatogonia, Cbx2[HA/HA], HA-ab9110

description:
CUT&RUN library
HACBX2_HA-ab9110

value: Testes

value: HA (Abcam, Cat# ab9110)


title: exp2_ch5_GEX

description: 10x Genomics


title: R26


title: exp2_ch4_GEX

description: 10x Genomics

value: Blood 2.1, Liver 2.1, Blood 2.3, Liver 2.3, Blood 2.5, Blood 2.8


title: FACS-sorted cKIT(+) spermatogonia, WT, HA-16B12 (neg. ctrl)

description:
CUT&RUN library
WT_HA-16B12_NegCtrl

value: wild type


title: BF-LE-02-PG-PSO_all

value: male


title: exp2_ch3_GEX

description: 10x Genomics

value: mRNA


title: iMG, rep2

description:
iPSCModels_raw.txt
iPSCModels_TPM.txt

value: iPSC-microglia


title: iMG, rep1

description:
iPSCModels_raw.txt
iPSCModels_TPM.txt


title: exp2_ch2_GEX

description: 10x Genomics


title: R24


title: FACS-sorted cKIT(+) spermatogonia, WT, HA-ab9110 (neg. ctrl)

description:
CUT&RUN library
WT_HA-ab9110_NegCtrl


title: xMG-8weeks, rep3

data_processing:
ATAC libraries were trimmed to 30bp and were mapped to hg38 genome using bowtie2 (Langmead and Salzberg, 2012) with default parameters. HOMER (Heinz et al., 2010) tag directories were created for mapped samples. Peaks were called using HOMER’s findPeaks command with the following parameters: “-style factor -size 200 - minDist 200” for ATAC-seq experiments. Peaks were merged with HOMER’s mergePeaks and annotated using HOMER’s annotatePeaks.pl using all tag directories.
Assembly: hg38
Supplementary files format and content: tab-delimited text files of peak files per each sample

extract_protocol:
For iMGs, cells were carefully manually scraped from the Matrigel coated plate and were concentrated by a 300 rcf x 5 minutes. For oMGs, organoids were carefully collected, allowed to settle by gravity, and then dissociated mechanically in staining buffer (HBSS 1x with 1mM EDTA and 1% BSA) using a 2 ml polytetrafluoroethylene pestle. iMGs and oMGs were stained with the following Biolegend antibodies/dyes: CD64-APC (305014), CX3CR1 PCP-Cy5.5 (341614), CD14-488 (325610), CD11b-PE (301309), HLADR PE-Cy7 (307616), CD45-APC-CY7 (368516), and Zombie Violet (1:1000, Biolegend) for live/dead discrimination. xMGs were isolated and sorted at indicated time points following xenotransplantation. Briefly, mouse brains were homogenized as previously described by gentle mechanical dissociation (Gosselin et al., 2014) and stained with DAPI, CX3CR1-APC (Biolegend 149008), anti-human CD11b-PE (Biolegend 301306), anti-human CD45-FITC (Biolegend 368508). Microglia were purified with either a BD Influx  or FACS  AriaFusion.
Approximately 30,000-50,000 isolated microglia were lysed in 50 µl lysis buffer (10 mM Tris-HCl pH 7.5, 10 mM NaCl, 3 mM MgCl2, 0.1% IGEPAL, CA-630, in water). Resulting nuclei were centrifuged at 500 rcf for 10 minutes. Pelleted nuclei were resuspended in 50 µl transposase reaction mix (1x Tagment DNA buffer (Illumina 15027866), 2.5 µl Tagment DNA enzyme I (Illumina 15027865), and incubated at 37C for 30 min on a heat block. For isolations resulting in under 30,000 microglia, microglia directly placed in 50 µl transposase reaction mix, as indicated above and incubated for 37C for 30 min. DNA was purified with Zymo ChIP DNA concentrator columns (Zymo Research D5205), eluted with 11 µl of elution buffer, and amplified using NEBNext High-Fidelity 2x PCR MasterMix (New England BioLabs M0541) with the Nextera primer Ad1 (1.25 µM) and a unique Ad2.n barcoding primer (1.25 µM) for 8-12 cycles. Libraries were size-selected through gel excision for fragments that were 175-255 bp and single-end sequenced for 51 cycles on a HiSeq 4000 or NextSeq 500.

value: Xenotransplanted iPSC-microglia

value: 8weeks post-xenotransplantation


title: R25


title: R23

value: 7 weeks


title: FACS-sorted cKIT(+) spermatogonia, WT, IgG

description:
CUT&RUN library
WT_IgG

value: cKIT(+) differentiating spermatogonia

value: IgG (Abcam, Cat# ab37415)


title: FACS-sorted cKIT(+) spermatogonia, WT, CBX2-855

description:
CUT&RUN library
WT_CBX2-855

value: CBX2 (home-made, 855)


title: xMG-8weeks, rep2


title: xMG-8weeks, rep1


title: R22


title: xMG-12days, rep4

value: 12days post-xenotransplantation


title: SNRNP200 cytoplasmic stress eCLIP rep 1 (IP)

first: Brian

last: Yee

address: 9500 Gilman Dr

department: Health

institute: UCSD

description: SNRNP200_stress.reproducible_enriched_windows.tsv.gz.bed

data_processing:
Data was processed using the eCLIP pipeline and available at https://github.com/YeoLab/skipper
Unique Molecular Identifiers (UMIs) were extracted using fastp 0.11.5 (https://github.com/OpenGene/fastp)
Post-umi-extracted reads were trimmed for adapter sequences and barcode sequences (eCLIP samples)
Mapping was then performed against the full human genome (hg38) including a database of splice junctions with STAR (v 2.7.6) allowing up to 100 multimapped regions.
Reads were then PCR deduplicated using UMIcollaps (https://github.com/Daniel-Liu-c0deb0t/UMICollapse
Enriched “windows” (IP versus SM-Input) was called on deduplicated reads using a GC-bias aware beta-binomial model. Each window (~100 b.p.) were partitioned from Gencode v38 and associated with a specific type of genomic region (CDS, UTR, proximal introns near splice site… etc). Windows are filtered using FDR < 0.2 and only reproducible windows between two replicates were used.
Data was processed using the eCLIP pipeline and available at https://github.com/YeoLab/skipper
Assembly: hg38
Supplementary files format and content: bed files contain Skipper windows.

source_name: Human epithelial cell

growth_protocol: DMEM + glutamine + 10% FBS

extract_protocol:
Cells were lysed and and sonicated in eCLIP lysis buffer, and RNA was fragmented using RNase I (Ambion) and RBP:RNA complexes were immunoprecipitated with RBP-specific antibody (A301-902A)
RNA was reverse transcripted with Superscript III, and adaptor was ligated with T4 RNA Ligase 1, then libraries were constructed with Q5 High-Fidelity 2x Master Mix (NEB)

treatment_protocol: 500 µM of arsenic

value: Human epithelial cell

value: Human epithelial kidney

value: 500 µM of arsenic

tag: fraction

value: IP


title: SNRNP200 cytoplasmic stress eCLIP rep 2 (IP)


title: SNRNP200 whole cell stress eCLIP rep2 (IP)

description: SNRNP200_HEKstress.reproducible_enriched_windows.tsv.gz.bed


title: xMG-12days, rep3


title: exp2_ch1_GEX

description: 10x Genomics

tag: donor

value: Blood and liver

value: MAIT and Tmem cells


title: PTEN null CD8T scRNA-seq

city: Beijing

first: Zihan

last: Xu

address: No.5 Yiheyuan Road Haidian District

institute: Peking University

data_processing:
For ATAC-seq and RNA-seq data, quality assessment of raw reads was achieved with FastQC and contaminants were removed using Trimgalore with parameters ‘-q 30 --length 30 --stringency 5’.
ATAC-seq: The clean paired-end reads were aligned to the mm10 reference genome by Bowtie2 with the parameters -D 20 -R 3 -N 0 -L 20 -i S,1,0.50. All unmapped reads, reads mapped to the mitochondrial genome, PCR duplicates and nonuniquely mapped reads were discarded. Peaks were called using the above filtered reads by MACS with the parameters --nolambda–nomodel.
RNA-seq: The clean paired-end reads were aligned to the mm10 reference genome by Tophat with the parameter --no-novel-juncs. Gene expression levels and differentially expressed genes between samples were calculated with Cuffdiff and visualized with the R package cummeRbund.
For scRNA-seq data, sequencing adapters, low quality sequences and amplification primers were trimmed from the raw sequencing reads. TopHat was applied to align clean reads to the mm10 reference genome. Uniquely aligned reads were counted by the tool ‘htseq-count’ of HTSeq.
Genome_build: mm10
Supplementary_files_format_and_content: peak text files
Supplementary_files_format_and_content: text files include FPKM values for each sample
Supplementary_files_format_and_content: text files include UMI counts for each single cell

source_name: thymus

extract_protocol:
Single cells were sorted into 96-well plates with lysis buffer.
Single cell RNA-seq barcoded libraries were generated and sequenced as previously described (Li et al., 2017)

value: CD8+ T

value: Ptennull


title: PTEN null prepro-B scRNA-seq Rep2

source_name: bone marrow

value: prepro-B


title: PTEN null prepro-B scRNA-seq Rep1


title: R19

value: RPE

value: WT

tag: time


title: SNRNP200 whole cell stress eCLIP rep1 (IP)


title: R20


title: FACS-sorted cKIT(+) spermatogonia, WT, H3K27me3, rep3

first: Jongmin

last: Kim

address: 930 Campus Road

institute: Cornell University

description:
CUT&RUN library
WT_H3K27me3_rep3

value: Testes

value: wild type

value: H3K27me3 (CST, clone C36B11, Cat# 9733)


title: xMG-12days, rep2

value: Xenotransplanted iPSC-microglia


title: PTEN null CD4T scRNA-seq

value: CD4+ T


title: R18

value: 5 weeks


title: xMG-12days, rep1

value: 12days post-xenotransplantation


title: PTEN null DP scRNA-seq

value: DP T


title: R17


title: CAPRIN eCLIP rep 1 (IP)

description: CAPRIN.reproducible_enriched_windows.tsv.gz.bed

value: Human epithelial cell

value: Human epithelial kidney


title: CAPRIN eCLIP rep 2 (IP)

value: IP


title: R21

value: 7 weeks


title: xMG-7days, rep3

value: 7days post-xenotransplantation


title: PTEN null DN4 scRNA-seq

value: DN4 T

value: Ptennull


title: G3BP1 eCLIP rep 2 (IP)

description: KR_G3BP1_HEK.reproducible_enriched_windows.tsv.gz.bed


title: xMG-7days, rep2


title: PTEN null DN3 scRNA-seq

value: DN3 T


title: R15

value: RPE

value: WT


title: G3BP1 eCLIP rep 1 (IP)


title: R14

value: 4 weeks


title: PTEN null DN2 scRNA-seq

value: DN2 T


title: SNRNP200 cytoplasmic eCLIP rep 2 (IP)

description: SNRNP200_cyto.reproducible_enriched_windows.tsv.gz.bed