title: CH02_2 scRRBSseq

description: genomic DNA digested with MspI + HaeIII and treated with bisulfite

data_processing:
scRNA data was aligned with STAR (v2.5.2) using twopassMode, gene level counts were generated using featureCounts (subread v1.5.2) with default parameters. The number of mutant and WT reads per cell was counted using a pileup at the mutation site (pySAM v0.8.2.1). Only reads with MAPQ > 40 and a specified minimum base quality at the mutation site were considered.
For scDNAme, each pool of 96 cells was first demultiplexed by Illumina i7 barcodes, resulting in four pools of 24 cells. Each pool of 24 cells was further demultiplexed by unique cell barcodes. Reads were assigned to a given cell if they matched 80% of the template adapters. Adapters and adapter reverse complements (6 bp) were trimmed from the raw sequence reads. After adapter removal, reads were trimmed by 5 bp from their 3’ end. We aligned trimmed reads to the hg38 human genome assembly using Bismark117 (v.0.14.5; parameters: -multicore 4 -X 1000 --un –ambiguous) running on bowtie2-2.2.8 aligner118. Bismark methylation extractor (--bedgraph --comprehensive) was used to determine the methylation state of each individual CpG. For downstream analyses, a site was considered methylated or unmethylated only if there was 90% agreement of the methylation state for all reads mapped to the site. Cells with coverage of at least 25,000 unique CpGs and bisulfite conversion rates of at least 99% were retained for downstream analyses.
Genome_build: hg38
Supplementary_files_format_and_content: Text files include  counts overlapping with annotated genes for all cells. Excel and comma-separated values files contain the filename conversion key to match scRNA with scDNAme cells, the sequencing summary statistics for each cell, and genotype call.
Supplementary_files_format_and_content: all_CpG_meth.csv: CpG by cell matrix with methylation values for all detected CpGs
Supplementary_files_format_and_content: scRNA_filename_key_to_scDNAme.csv: Filename conversion key to match scRNA with scDNAme cells
Supplementary_files_format_and_content: scDNAme_cell_metadata.xlsx: Summary statistics and metadata of all cells
Supplementary_files_format_and_content: scRNA_cell_metadata.xlsx: Summary statistics and metadata of all cells
Supplementary_files_format_and_content: gene_exp_mtx.txt: Gene count matrix from single-cell RNA-seq


title: GB, N8mg/L, AcuteExp1mg/L, 3

status: Public on Jul 05 2022

type: SRA

city: Johnson City

first: Lev

middle: Y

last: Yampolsky

state: Tennessee

address: 811 Lazywood Dr

department: Biological sciences

country: USA

institute: East Tennessee State University

library_source: transcriptomic

data_processing:
Basecalling: ONT guppy_basecaller
Demultiplexing, trimming: ONT guppy_barcoder
Alignment to reference transcriptome: ONT guppy_aligner to longest transcript of DM3 reference (BioProject ID: PRJNA624896)
Merging and indexing BAM files, exporting read counts: samtools
Filtering: retain transcripts with >=72 reads total
Assembly: N/A
Supplementary files format and content: Hyp84AC.readCountsRPKM.txt (read counts and RPKMs in tab-separated text file)

molecule: total RNA

organism: Daphnia magna

source_name: Whole body homogenates, 2 individuals pooled

growth_protocol: 20 oC, density 1 ind.20 mL, food 100,000 Scenedesmus cells/mL/day; 25 days; either at normoxia (8 mg O2) or mild intermittent hypoxia (4 mg/L O2 twice daily)

extract_protocol:
Qiagen RNAeasy kit (Cat ID: 74134) with DNAse treatment; Qubit quantification
ONT PCR-cDNA Barcoding kit protocol (SQK-PCB109)

treatment_protocol: either no treatment (Control), or 12 h at acute severa hypoxia (<1 g/L O2)

tag: tissue

value: Whole body

tag: clone

tag: chronic hypoxia

value: normoxia 8mgO2/L

tag: acute hypoxia

tag: Sex

value: female


title: ILC3, nonstimulated, biol rep3

city: Rehovot

first: Jakub

last: Abramson

address: 234 Herzl st.

department: Department of Immunology

country: Israel

institute: Weizmann Institute of Science

data_processing:
Data was processed using UTAP pipeline (Kohen et al., 2019). Bcl files were converted into fastq by bcl2fastq. Demultiplexing is done using the sample barcode found on R2, the UMI found on R2 is inserted in the read name (in R1 fastq).  R1 Fastq data submitted.
Reads were trimmed using cutadapt and mapped to genome (mm10) STAR v2.4.2a (default parameters).
The pipeline quantifies the 3’ of Gencode annotated genes (The 3’ region contains 1,000 bases upstream of the 3’ end and 100 bases downstream). Counting proceeded over genes annotated in RefSeq release using htseq-count (union mode). Genes having minimum 5 UMI-corrected reads in at least one sample, were considered.
Differential expression analysis was performed using DESeq2 with the betaPrior set to False. Raw P values were adjusted for multiple testing using the procedure of Benjamini and Hochberg.
Assembly: mm10
Supplementary files format and content: tab delimited files include raw counts values for each sample, normalized abundance measurements and DESeq2 analysis

organism: Mus musculus

source_name: lymph nodes

extract_protocol:
Lymph nodes were collected and subjected to several rounds of enzymatic digestion by Dispase I. Single cell suspension was depleted of lineage positive cells using LS-column based MACS enrichment by cocktail of biotinylated antibodies (TCR-β, CD3, CD19, B220, CD11b, F4/80, Gr1, CD11c) and anti-biotin microbeads. T-cells were isolated by meshing the skin-draining lymph nodes and spleens through 40 μm nylon mesh. Single cell suspension was subjected to flow cytonetry based sorting.
Single cell suspensions were directly FACS sorted to Lysis/Binding buffer (Invitrogen) and frozen on dry ice. RNA was isolated using Dynabeads (Invitrogen) according to manufactures protocol. The MARS-seq protocol described elsewhere was followed to generate the sequencing libraries (Jaitin D.A. et al., Science, 2014)
RNA sequencing 3' end with bulk MARS-Seq

value: lymph nodes

tag: strain

tag: cell type

tag: treatment

value: nonstimulated


title: GBM SF10099_snRNA

city: San Francisco

first: Aaron

last: Diaz

state: CA

department: Neurological Surgery

institute: University of California, San Francisco

description: Single Nuceli RNA-Seq

data_processing:
We utilized CellRanger (version 3.0.2) for data pre-processing and gene expression quantification, following the guidelines from the CellRanger website (https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/advanced/references#premrna).
The CellRanger ATAC software (version 1.1.0) was used for read alignment, deduplication, and identifying transposase cut sites.
The presence/absence of somatic CNVs was assessed with CONICSmat. ELSA was used for cell type classification.
Genome_build: hg38
Supplementary_files_format_and_content: SnRNA-seq: feature read count matrix (.txt)  files.   Includes cell barcodes, genes and count. SnATAC-seq: peak, matrix, barcode file.

organism: Homo sapiens

source_name: Brain

extract_protocol:
Nuclei were extracted from frozen tissues following the “Frakenstein” protocol developed by Luciano Martelotto, Ph.D., Melbourne, Centre for Cancer Research, Victorian Comprehensive Cancer Centre, and available from 10X Genomics
For snRNA-seq(frozen tissues), ~15,000 nuclei were loaded per capture. Single-nucleus capture, reverse transcription, cell lysis, and library preparation were performed per manufacturer’s protocol. Sequencing was performed on an Illumina NovaSeq using a paired-end 100bp protocol.

tag: progression

value: Primary

tag: diagnosis

value: GBM

tag: age

tag: gender

value: Male

tag: pair#


title: CH04_2 scRRBSseq

city: New York

first: Neville

last: Dusaj

state: NY

institute: Weill Cornell Medicine

library_source: genomic

molecule: genomic DNA

source_name: GCSF mobilized stem cells from patient

extract_protocol:
Cryopreserved patient samples approved by the Institutional Review Board of Dana Farber Cancer Institute were thawed and stained using standard procedures (10 min, 4°C) with the surface antibody CD34-PE-Vio770 (clone AC136, Miltenyi Biotec) and DAPI (Sigma-Aldrich). Cells were then sorted for DAPI-negative, CD34+ and CD34-negative cells using BD Influx.
Methylome and whole-transcriptome library preparation and sequencing were performed as previously described. Briefly, genomic DNA (gDNA) and mRNA have been separated manually. A modified oligo-dT primer (5′-biotin-triethyleneglycol-AAGCAGTGGTATCAACGCAGAGTACT30VN-3′, where V is either A, C or G, and N is any base; IDT) was conjugated to streptavidin-coupled magnetic beads (Dynabeads, Life Technologies) according to the manufacturer’s instructions. To capture polyadenylated mRNA, we added the conjugated beads (10 μl) directly to the cell lysate and incubated them for 20 min at room temperature with mixing to prevent the beads from settling. The mRNA was then collected to the side of the well using a magnet, and the supernatant, containing the gDNA, was transferred to a fresh plate. Single-cell complementary DNA was amplified from the tubes containing the captured mRNA according to a variation of the Smart-Seq2 protocol using molecular crowding to increase sensitivity. After amplification and purification using 0.8X SPRI beads, 0.5ng cDNA was used for Nextera Tagmentation and library construction. Library quality and quantity were assessed using Agilent Bioanalyzer 2100 and Qubit, respectively. Genomic DNA present in the pooled supernatant and wash buffer from the mRNA isolation step was concentrated on 0.8X SPRI beads and eluted directly into the reaction mixtures for Msp1(+/- HaeIII) (Fermentas) enzymatic reaction (10mL final reaction) for 90 min at 37°C. Heat-inactivation was performed for 10 min at 70°C. Digested DNA was filled-in and A-tailed at the 3’ sticky ends in 8.5 mL final volume of 1X CutSmart with 2.5 units of Klenow fragment (Exo-, Fermentas). Reaction was supplemented with 1 mM dATP and 0.1 mM dCTP and 0.1 mM dGTP (NEB) and performed as follows in a thermocycler: 30°C for 25 min, 37°C for 25 min and heat-inactivation at 70°C for 10 min. Custom barcoded methylated adaptors (0.1 mM) were then ligated overnight at 16°C with the dA-tailed DNA fragments in the presence of 800 units of T4 DNA ligase (NEB) and 1 mM ATP (Roche) in a final volume of 11.5 mL of 1X CutSmart buffer. T4 DNA ligase heat-inactivation was performed at 70°C for 15 min the next day. Genomic DNA from 24 individual cells were pooled together according to their barcodes, giving, for a 96-well plate, 4 pools of 24 cells. Pooled genomic DNA was cleaned-up and concentrated using 1.8X SPRI beads (Agencourt AMPure XP - Beckman Coulter). Each pool was then sodium bisulfite converted (Fast Epitect Bisulfite, Qiagen) following manufacture recommendations or converted using EM-seq kit (New Englands Biolabs) following manufacture recommendations. Converted DNA was then amplified using primers containing Illumina i7 and i5 index. Following Illumina pooling guidelines, a different i7 index was used for every 24-cell pool, allowing multiplexing of 96 cells for sequencing on one Illumina HiSeq lane. Library enrichment was done using KAPA HiFi Uracil+ master mix (Kapa Biosystems) and the following PCR condition was used: 98°C for 45 secs; 6 cycles of: 98°C for 20 secs, 58°C for 30 secs, 72°C for 1 min; followed by 12 cycles of: 98°C for 20 secs, 65°C for 30 secs, 72°C for 1 min. PCR was terminated by an incubation at 72°C for 5 min. Enriched libraries were cleaned-up and concentrated using 1.3X SPRI beads. DNA fragments between 200 bp and 1 Kb were size-selected and recovered after resolving on a 3% NuSieve 3:1 agarose gel. Libraries molarity concentration calculation was obtained by measuring concentration of double stranded DNA (Qubit) and quantifying the average library size (bp) using an Agilent Bioanalyzer. Every 24-cells pool was mixed with the others pool in an equimolar ratio. All cells from a 96-well plate were sequenced as paired-end on HiSeq 2500 with 10% PhiX spike-in. Negative controls (empty wells with no cells) were used to control for non-specific amplification of the libraries. Single cell bisulfite-seq or enzymatic methyl-seq (reduced representation) and single-cell RNA-seq (full-length transcriptome profiling)
Single cell bisulfite-seq or enzymatic methyl-seq (reduced representation) and single-cell RNA-seq (full-length transcriptome profiling)

value: Blood

tag: disease state

value: Clonal hematopoiesis


title: ILC3, nonstimulated, biol rep2


title: KO 0-3 Liver

city: irvine

first: Siwei

last: Chen

address: 66304 Verano Road South

department: Computer Science

institute: University of California, Irvine

data_processing:
Alignment to reference assembly mm10 and known splice junctions extracted from the RefSeq database performed using Tuxedo pipeline tophat
Read counting for each gene and normalization to FPKM values  using Tuxedo pipeline
Genome_build: mm10
Supplementary_files_format_and_content: Gene expression results FPKM values) of the standard RNAseq analysis are reported in the genes_fpkm.csv file

source_name: liver

extract_protocol: Standard Illumina RNA-seq protocol

tag: genotype/variation

value: KO

tag: time point


title: DC, nonstimulated, biol rep3

value: DC


title: DC, stimulated, biol rep3

value: Candida albicans


title: CH04_2 scRNAseq + Genotyping

description: polyA mRNA

molecule: polyA RNA


title: GB, H4mg/L, AcuteExp1mg/L, 6

value: hypoxia 4mgO2/L


title: FI, N8mg/L, AcuteExp1mg/L, 3


title: FI, N8mg/L, Control, 8

value: Control


title: KO 0-2 Liver


title: FI, N8mg/L, Control, 7


title: WT 0-2 Liver

value: WT


title: CH04_1 scRREMseq

description: genomic DNA digested with MspI and treated with TET2/APOBEC

data_processing:
Library strategy: enzymatic methyl-seq (reduced representation)
scRNA data was aligned with STAR (v2.5.2) using twopassMode, gene level counts were generated using featureCounts (subread v1.5.2) with default parameters. The number of mutant and WT reads per cell was counted using a pileup at the mutation site (pySAM v0.8.2.1). Only reads with MAPQ > 40 and a specified minimum base quality at the mutation site were considered.
For scDNAme, each pool of 96 cells was first demultiplexed by Illumina i7 barcodes, resulting in four pools of 24 cells. Each pool of 24 cells was further demultiplexed by unique cell barcodes. Reads were assigned to a given cell if they matched 80% of the template adapters. Adapters and adapter reverse complements (6 bp) were trimmed from the raw sequence reads. After adapter removal, reads were trimmed by 5 bp from their 3’ end. We aligned trimmed reads to the hg38 human genome assembly using Bismark117 (v.0.14.5; parameters: -multicore 4 -X 1000 --un –ambiguous) running on bowtie2-2.2.8 aligner118. Bismark methylation extractor (--bedgraph --comprehensive) was used to determine the methylation state of each individual CpG. For downstream analyses, a site was considered methylated or unmethylated only if there was 90% agreement of the methylation state for all reads mapped to the site. Cells with coverage of at least 25,000 unique CpGs and bisulfite conversion rates of at least 99% were retained for downstream analyses.
Genome_build: hg38
Supplementary_files_format_and_content: Text files include  counts overlapping with annotated genes for all cells. Excel and comma-separated values files contain the filename conversion key to match scRNA with scDNAme cells, the sequencing summary statistics for each cell, and genotype call.
Supplementary_files_format_and_content: all_CpG_meth.csv: CpG by cell matrix with methylation values for all detected CpGs
Supplementary_files_format_and_content: scRNA_filename_key_to_scDNAme.csv: Filename conversion key to match scRNA with scDNAme cells
Supplementary_files_format_and_content: scDNAme_cell_metadata.xlsx: Summary statistics and metadata of all cells
Supplementary_files_format_and_content: scRNA_cell_metadata.xlsx: Summary statistics and metadata of all cells
Supplementary_files_format_and_content: gene_exp_mtx.txt: Gene count matrix from single-cell RNA-seq


title: KO 0-1 Liver


title: DC, nonstimulated, biol rep2


title: CH02_1 scRNAseq + Genotyping

description: polyA mRNA


title: CH04_1 scRNAseq + Genotyping

description: polyA mRNA


title: WT 0-1 Liver


title: FI, N8mg/L, AcuteExp1mg/L, 1


title: CH03 Amplicon

data_processing:
10x data was processed using Cell Ranger 3.0.1 using default parameters. RNA reads were aligned to the human or mouse referece genome, or human and mouse reference concatenation for species mixing experiment.
Amplicon reads was processed following steps: 1) Identification of reads with proper priming, 2) Identification of cell barcodes (CB) that match with the whitelisted CB, 3) Replacement of CB that are not perfectly matched with the whitelist CB, 4) Deduplication of reads, 5) Analyze reads with CB that are also observed in the 10x scRNA-seq data, 6) Collapse networks of closely related UMIs within CBs to correct for sequencing error, 7) Matching of CB/UMI pairs found in 10x scRNA-seq data, discarding those matching non-DNMT3A genes and those matching no gene below a supporting read threshold.
Genome_build: hg19
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 three columns: barcodes (BC), number of wildtype calls (num.WT.call), and number of mutant calls (num.MUT.call).

extract_protocol:
Cryopreserved patient samples approved by the Institutional Review Board of Dana Farber Cancer Institute were thawed and stained using standard procedures (10 min, 4°C) with the surface antibody CD34-PE-Vio770 (clone AC136, Miltenyi Biotec) and DAPI (Sigma-Aldrich). Cells were then sorted for DAPI-negative, CD34+ and CD34-negative cells using BD Influx.
The standard 10x Genomics Chromium v.3 protocol was carried out according to manufacturer’s recommendations (10x Genomics, Pleasanton, CA) until after emulsion breakage and recovery of first strand cDNA. A gene-specific DNMT3A primer was spiked into 10x primer mix at 1% of the concentration of the cDNA amplification primers for the initial cDNA PCR step. Then, during the amplification step, the 10x cDNA library underwent an extra cycle of PCR beyond the manufacturer’s recommended number of cycles. After cleanup with SPRIselect, a small portion of the cDNA library, 3 µL (~10% of total) was aliquoted for targeted genotyping, and the remaining cDNA underwent the standard 10x protocol. The cDNA set aside for GoT was amplified for 3 to 4 additional cycles using KAPA HiFi HotStart ReadyMix (KAPABiosystems) and 10x primer mix to provide sufficient material for the enrichment step. After clean-up, locus-specific reverse primers and the generic forward SI-PCR were used to amplify the site of interest of the cDNA template using ~10 PCR cycles. The locus-specific reverse primers contain a partial Illumina read 2 handle, a stagger to increase the complexity of the library for optimal sequencing and a gene specific region to allow specific priming. The SI-PCR oligo (10x) anneals to the partial Illumina read 1 sequence at the 3’ end of the molecule, preserving the cell barcode (CB) and UMI. After the initial amplification and SPRI purification to remove unincorporated primers, a second PCR was performed with a generic forward PCR primer (P5_generic) 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. The targeted amplicon library was subsequently sequenced separately. The cycle settings were as follows: 28 cycles for read 1, 98 or 130 cycles for read 2, and 8 cycles for sample index.
Single-cell RNA-seq (10X) with targeted amplification of a mutation of interest.


title: 141336_KO

city: Ann Arbor

first: Michael

middle: Kemper

last: Scales

state: MI

address: 109 Zina Pitcher Place

department: Cell and Developmental Biology

institute: University of Michigan

data_processing:
Sequenced on a NovaSeq 150-PairedEnd s4 flowcell, RecipeVersion 1.6.0, RtaVersion 3.4.4.
STAR 2.7.2b was used to align reads with the following parameters --outFilterMultimapNmax 20 --alignSJoverhangMin 8 --alignSJDBoverhangMin 1 --outFilterMismatchNmax 999 --outFilterMismatchNoverReadLmax .04 --alignIntronMin 20 --alignIntronMax 1000000 --alignMatesGapMax 1000000
featureCounts from the Bioconductor Rsubread package was used to count features from gencode.vM23.annotation.gtf.
Linear models calculated using standard limma pipeline and the voom technique.
Genome_build: GRCm38.p6
Supplementary_files_format_and_content: RawCounts_matrix.txt: Tab-delimited text file with 3 columns of annotation followed by 6 columns of counts, 1 for each sample.
Supplementary_files_format_and_content: Voom_LogCPM.txt: Tab-delimited text file with 3 columns of annotation followed by 6 columns of logcpm values from limma's voom, 1 for each sample.

source_name: Pancreatic Fibroblasts

growth_protocol: Fibroblasts were grown to confluence in three independent 6-well tissue culture wells per experimental group in DMEM (Invitrogen, Gibco) + 10% Calf Serum + 1% Pen/Strep.

extract_protocol:
Cells were lysed in Qiagen Buffer RLT and RNA was isolated using a Qiagen RNeasy plus Mini Kit (Catalog # 74134). Samples were treated with DNase I (Qiagen 79254) to remove any contaminating DNA. 9 micrograms of RNA was then submitted to generate RNA libraries.
RNA libraries were prepared for sequencing using Poly-A NEB ultra 2 kit from 250ng input and 13 cycles PCR.

value: Immortalized pancreatic fibroblasts

value: Mixed

tag: genotype


title: DC, stimulated, biol rep2

tag: tissue


title: WT 0-3 Liver


title: FI, H4mg/L, Control, 12

value: Whole body

tag: clone


title: Aire-GFP+ILC3, stimulated, biol rep3


title: FI, N8mg/L, AcuteExp1mg/L, 2

tag: chronic hypoxia

value: normoxia 8mgO2/L

value: female


title: CH02 Amplicon


title: CH02_2 scRNAseq + Genotyping

description: polyA mRNA


title: 141335_KO


title: 141334_KO


title: FI, H4mg/L, Control, 11

tag: acute hypoxia


title: Aire-GFP+ILC3, stimulated, biol rep1

value: lymph nodes


title: DC, nonstimulated, biol rep1

value: nonstimulated


title: CH01 Amplicon


title: FI, H4mg/L, Control, 10

tag: Sex


title: CH03 10X


title: FI, H4mg/L, AcuteExp1mg/L, 6


title: CH02 10X


title: 141330_WT

tag: strain


title: 141329_WT

tag: cell type


title: Aire-GFP+ILC3, nonstimulated, biol rep1

tag: treatment


title: Aire-GFP+ILC3, nonstimulated, biol rep2


title: 141328_WT


title: FI, H4mg/L, AcuteExp1mg/L, 4


title: CH01 10X


title: Aire-GFP+ILC3, stimulated, biol rep2


title: pDCs CpG+desialylated IVIG stimulation rep 3

status: Public on Jul 04 2022

city: Shanghai

first: Haibo

last: Zhou

address: No.160, Pujian Road

department: Department of Rheumatology

country: China

institute: Renji Hospital

data_processing:
Illumina Casava1.7 software used for basecalling.
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence, then mapped to mm10 whole genome using HISAT2 (Kim et al., 2015;Pertea et al., 2016).
Reads Per Kilobase of exon per Megabase of library size (RPKM) were calculated using StringTie(Pertea et al., 2016).
Assembly: mm10
Supplementary files format and content: tab-delimited text files include normalized counts for each Sample ...

source_name: peripheral blood

growth_protocol: Cells were cultured in RPMI 1640 medium (Gibco) with 10% fetal bovine serum (FBS, Gibco) and 1% Penicillin-Streptomycin (Gibco) at 37°C and 5% CO2.

extract_protocol:
Total RNA was extracted by Trizol reagent (Invitrogen) and determined by Bioanalyzer 4200 (Agilent, Santa Clara, CA, USA).
Next-generation libraries of mRNA-seq were prepared using VAHTS mRNA-seq v2 Library Prep Kit for Illumina® (Vazyme, Nanjing, China). The Library quality was determined by Bioanalyzer 4200 (Agilent, Santa Clara, CA, USA).

treatment_protocol: Cells were preincubated with or without 10mg/ml IVIG or desialylated IVIG, followed by stimulation with CpG overnight.

value: peripheral blood

value: plasmacytoid dendritic cells

value: CpG+desialylated IVIG


title: pDCs CpG+desialylated IVIG stimulation rep 1


title: pDCs CpG+desialylated IVIG stimulation rep 2


title: DC, stimulated, biol rep1


title: pDCs CpG+IVIG stimulation rep 1

value: CpG+IVIG


title: pDCs CpG+IVIG stimulation rep 3


title: Aire-GFP+ILC3, nonstimulated, biol rep3


title: pDCs CpG stimulation rep 1

value: CpG


title: pDCs CpG stimulation rep 3


title: SFB PAR-Clip

city: Hangzhou

first: Xiaochun

last: Yu

state: Zhejiang

address: 18 Shilongshan Street

institute: Westlake University

data_processing:
The gene annotation (GTF format) of CHM13 is generated with parameters: gffread chm13.draft_v1.1.gene_annotation.v4.gff3 -o chm13.draft_v1.1.gene_annotation.v4.gtf
The STAR reference of human CHM13 genome assembly v1.1 is built with parameters: STAR --runThreadN 8 --runMode genomeGenerate --genomeDir hsa_chm13v1.1_genanno_v4 --genomeFastaFiles chm13.draft_v1.1.fasta --sjdbGTFfile chm13.draft_v1.1.gene_annotation.v4.gtf --sjdbOverhang 150
The BLAST database of human ribosomal DNA is built with parameters: makeblastdb -in Hsa_rDNA_Kim.fa -dbtype nucl -out HsaKimrDNA -parse_seqids
RNA-seq reads are aligned to the pre-built human CHM13 v1.1 using STAR version 2.7.6a with the following parameters: STAR --runMode alignReads --genomeLoad NoSharedMemory --runThreadN 8 --readFilesCommand gunzip -c --genomeDir [dir_of_hsa_CHM13v1.1] --readFilesIn [read1.fq.gz] [read2.fq.gz] --chimOutJunctionFormat 1 --outReadsUnmapped Fastx --chimSegmentMin 12 --chimJunctionOverhangMin 12 --chimSegmentReadGapMax 3 --alignSJDBoverhangMin 10 --alignMatesGapMax 100000 --alignIntronMax 100000 --alignSJstitchMismatchNmax 5 -1 5 5 --twopassMode Basic --outFileNamePrefix [outdir_bam] --sjdbOverhang 150 --outSAMstrandField intronMotif --outSAMattributes NH HI NM MD AS XS --outSAMunmapped Within --quantMode TranscriptomeSAM GeneCounts --outSAMtype BAM SortedByCoordinate --limitBAMsortRAM 50000000000 --outFilterMultimapNmax 50
Gene count data is generated using featureCounts version 2.0.2 with the following parameters: featureCounts -T 8 -p -t exon -g gene_id -a [location_of_hsa_CHM13v1.1_gtf] -o [output_featurecounts.txt] [star_align_output_sorted.bam]
The unaligned reads are converted to FASTA format and the reads shorter than 18nt are excluded using seqkit version 2.1.0: seqkit fq2fa -j 16 [unmapped_reads] | seqkit seq -m 18 -o [unmapped_reads_filtered.fa]
The unaligned sequences are aligned with human ribosomal DNA sequence (KY962518.1) using BLAST 2.11.0+ with following parameters: blastn -db [path_of_blastdb_HsaKimrDNA] -query [unmapped_reads_filtered.fa] -outfmt '6 qseqid sseqid pident length slen qlen mismatch gapopen qstart qend sstart send evalue bitscore' -out [blast_results_table]
The BLAST data are filtered, only results in 45S region are kept. These results were splitted into bins of 10nt.
Assembly: human CHM13 v1.1, and the reference sequence of human ribosomal DNA is extracted from NCBI (GenBank: KY962518.1).
Supplementary files format and content: FASTA (reference sequence of Human ribosomal DNA)
Supplementary files format and content: TXT (readpergene table generated from STAR)
Supplementary files format and content: TXT (featurecounts table generated from featurecounts)
Supplementary files format and content: TXT (10nt binned blast results)

source_name: Embryonic kidney

growth_protocol: 293T cells stably expressing SFB empty vector, BRCA1 BRCT or BARD1 cells were incubated with DMEM in 37℃，5% CO2 incubator.

extract_protocol:
Cells were incubated with 0.1 mM of 6SG for overnight followed by 0.4 Jcm2 365nm wavelength UV treatment.RNA were isolated with PAR-Clip by streptavidin resin and Flag resin.
Library preparation and sequencing were finished by Geneseed Technology CO., Ltd., Guangzhou, China. The protocol is as follows: 1) RNA quality and RNA sample assessment; 2) RNAs were fragmented, add adapters and indexes; 3) PCR; 4)Separation and cyclization; 5) Accurate quantification; 6) Sequencing.

treatment_protocol: PAR-Clip extract BARC1/BARD1 assiciated RNA

value: Embryonic kidney

tag: cell line

value: SFB empty vector


title: FI, H4mg/L, AcuteExp1mg/L, 5

value: hypoxia 4mgO2/L


title: BRCA1 BRCT PAR-Clip

value: BRCA1 BRCT

value: PAR-Clip by streptavidin resin and Flag resin


title: SLAM-seq_S9CC13_R6_3h

city: Stanford

first: Sahin

last: Naqvi

state: CA California

address: 265 Campus Drive, Lokey Stem Cell Research Building/SIM1

institute: Stanford University

data_processing:
Lexogen adapter sequences and low-quality bases were trimmed from sequencing reads (read 1 only) using skewer, followed by trimming of poly(A) sequences. Trimmed reads were used as input to slamdunk v0.4.3 (Neumann et al., 2019), with the following individual step parameters modified from default: map, -n 100 -5 0; count, -l 150.
Assembly: hg38
Supplementary files format and content: Total or T-C conversion-containing read counts over annotated 3' UTR regions
Library strategy: SLAM-Seq

source_name: H9 hESC

growth_protocol: hESCs were differentiated to cranial neural crest cells (CNCCs) using a protocol described previously (Bajpai et al., 2010; Prescott et al., 2015). Briefly, hESCs were grown for 5-6 days until large colonies formed, then were disaggregated using collagenase IV and gentle pipetting. Clumps of ~200 hESCs were washed in PBS and transferred to a 10cm Petri dish in neural crest differentiation media (NDM). NDM: 1:1 ratio of DMEM-F12 and Neurobasal, 0.5x Gem21 NeuroPlex Supplement With Vitamin A (Gemini, 400-160), 0.5x N2 NeuroPlex Supplement (Gemini, 400-163), 1x antibiotic/antimycotic, 0.5x Glutamax, 20ng/ml bFGF (PeproTech, 100-18B), 20ng/ml EGF (PeproTech, AF-100-15) and 5ug/ml bovine insulin (Gemini Bio-Products, 700-112P). After 7-8 days, neural crest emerged from neural spheres attached to the Petri dish, and after 11 days, neural crest cells were passaged onto fibronectin-coated 6-well plates (~1m cells/well) using accutase and fed with neural crest maintenance media (NMM). NMM: 1:1 ratio of DMEM-F12 and neurobasal, 0.5x Gem21 NeuroPlex Supplement with Vitamin A (Gemini, 400-160), 0.5x N2 NeuroPlex Supplement (Gemini, 400-163), 1x antibiotic/antimycotic, 0.5x Glutamax, 20ng/ml bFGF, 20ng/ml bFGF EGF and 1mg/ml BSA (Gemini). After 2-3 days, neural crest cells were plated at ~1m cells/well of a 6-well plate and the following day cells were fed with neural crest long-term media. Long term media: neural crest maintenance media + 50pg/ml BMP2 (PeproTech, 120-02) + 3uM CHIR-99021 (Selleck Chemicals, S2924) (BCh media). After transition to BCh media, CNCCs at subsequent passages were plated at ~800,000 cells/well of a 6-well plate. CNCCs were then passaged twice to passage 4 where depletion experiments were performed

extract_protocol:
Total RNA was extracted using Trizol reagent, phenol-chloroform extraction was performed, and the aqueous phased was used as input to the Quick-RNA Miniprep kit. During RNA extraction with Quick-RNA Miniprep kit, 0.1 mM DTT was added to the the RNA wash and RNA pre-wash buffers, but the on-column DNase I step was skipped. RNA was eluted in H20 with 1 mM DTT, quantified with Qubit RNA Broad Range assay (ThermoFisher, Q10211), and > 2 ug total RNA was used as input to the alkylation reaction. Alkylation was performed in dark tubes after which light exposure was allowed, and after quenching RNA was purified and subjected to on-column DNase I digestion using the RNA Clean & Concentrator-5 kit (Zymo, R1013).
500 ng alkylated RNA was used as input to QuantSeq 3’ mRNA-Seq Library Prep Kit FWD with unique dual index (UDI) add-on (Lexogen, 113.96), with 15 cycles of PCR amplification. Library size distributions were confirmed by separation on a PAGE gel and staining with SYBRGold and pooled based on quantifications from Qubit dsDNA High Sensitivity Kit (ThermoFisher , Q32854).

treatment_protocol: dTAGV-1 (Tocris, 6914) at a range of concentrations was added to BCh media, with an equivalent amount of DMSO as vehicle control, for the indicated times. 4-Thiouridine (4SU) was incorporated into nascent transcripts by incubating CNCCs with BCh media containing 100 uM 4SU, as well as DMSO or 500 nM dTAGV-1 depending on experimental condition, for 2 hours. Plates were covered in foil and handling was done in a hood with no light where possible. For 3 and 24 hour depletion experiments, labeling was started at 1 and 22 hours after dTAGV-1 addition, respectively.

tag: differentiation batch

value: H9 hESC

value: hESC-derived P4 CNCC

tag: treatment concentration_(m)

tag: treatment time

tag: relative sox9_level


title: SLAM-seq_S9CC47_R5_3h


title: WT_Rpe_5en7

data_processing:
TruSeq adapter sequences and low-quality bases were trimmed from sequencing reads using skewer, and transcript levels were quantified using salmon v1.4.0 quant with the following settings: --gcBias --seqBias -l A.
Salmon abundance files were summarized to the gene level and imported into R with the tximport package v1.20.0 with countsFromAbundance = “lengthScaledTPM”.
Assembly: hg38
Supplementary files format and content: Counts and abundances (TPM) summarized to gene level with tximport

extract_protocol:
Total RNA was extracted from CNCCs using Trizol reagent (Invitrogen) followed by Quick-RNA Miniprep kit (Zymo) with on-column DNase I digestion.
Libraries were prepared with the NEBNext Ultra II RNA Library Prep Kit for Illumina (NEB #E7770S/L)

treatment_protocol: dTAGV-1 (Tocris, 6914) at a range of concentrations was added to BCh media, with an equivalent amount of DMSO as vehicle control, for the indicated times.

value: RPe

value: WT


title: WT_RPe_0


title: SLAM-seq_S9CC47_R6_0h


title: SLAM-seq_S9CC13_R6_0h


title: SLAM-seq_S9CC47_R6_24h


title: SLAM-seq_S9CC47_R6_3h


title: S9CC47_R6_1.6e-8M


title: SLAM-seq_S9CC47_R5_0h


title: SLAM-seq_S9CC13_R6_24h


title: pDCs CpG stimulation rep 2


title: S9CC47_R6_5e-9M


title: BARD1 PAR-Clip


title: S9CC47_R6_5e-7M


title: pDCs CpG+IVIG stimulation rep 2


title: S9CC47_R6_1.6e-9M


title: S9CC47_R6_5e-10M


title: S9CC47_R6_0M


title: S9CC47_R5_5e-7M


title: S9CC47_R5_1.6e-8M


title: S9CC47_R5_5e-9M


title: S9CC47_R5_5e-10M


title: S9CC47_R5_1.6e-9M


title: SLAM-seq_S9CC47_R5_24h


title: S9CC47_R5_0M


title: S9CC13_R6_5e-7M


title: S9CC13_R6_5e-9M


title: S9CC13_R6_1.6e-8M


title: S9CC13_R6_1.6e-9M


title: WT_RSh_5e-7M

value: RSh


title: S9CC13_R11_0M


title: S9CC13_R10_1.6e-8M


title: WT_R8_0M


title: WT_RSh_0M


title: S9CC13_R6_0M


title: WT_R8_5e-7M


title: S9CC13_R10_0M


title: S9CC13_R11_5e-7M


title: S9CC13_R11_5e-9M


title: S9CC13_R11_5e-10M


title: S9CC13_R11_1.6e-9M


title: S9CC13_R11_1.6e-8M


title: S9CC13_R10_5e-7M


title: S9CC47_R8_1.6e-8M


title: S9CC13_R10_1.6e-9M


title: S9CC13_R10_5e-10M


title: S9CC47_R8_5e-9M


title: S9CC47_R8_5e-7M


title: S9CC47_R8_5e-10M


title: S9CC47_R8_1.6e-9M


title: S9CC13_R8_1.6e-8M


title: S9CC13_R8_5e-7M


title: S9CC47_R8_0M


title: S9CC13_R8_1.6e-9M


title: S9CC13_R6_5e-10M


title: S9CC13_R8_5e-10M


title: S9CC13_R10_5e-9M


title: S9CC13_R8_0M


title: WT_R5_untr_input

data_processing:
Truseq adapter sequences and low-quality bases (-Q 10) were trimmed from sequencing reads using skewer v0.2.2 and aligned to the human genome (hg38) using bowtie2 v2.4.1 with the following settings: --very-sensitive, --X 2000. Read mate pair information was corrected with samtools v1.10 fixmate, PCR duplicates were removed using samtools markdup, and mitochondrial reads and low mapping quality reads (-q 20) were removed using samtools view.  bigWig files for visualization were generated using deeptools v3.5.0 bamCoverage with the following settings: -bs 10 --normalizeUsing RPGC --samFlagInclude 64 --samFlagExclude 8 --extendReads.
Reads were counted over reproducible ATAC peak regions using bedtools
Supplementary files format and content: H3K27ac ChIP-Seq read counts over reproducible ATAC peak regions
Supplementary files format and content: V5 ChIP-Seq bigWig coverage files

extract_protocol:
One fully confluent 10 cm plate of cells was cross-linked per ChIP experiment in 10 mL PBS with 1% methanol-free formaldehyde for 10 min and quenched with a final concentration of 0.125M glycine for 5 min with nutation. Cross-linked cells were scraped into tubes with 0.001% Triton X in PBS, washed with PBS without Triton, pelleted by centrifugation, flash-frozen in liquid nitrogen and stored at −80°C. Samples were defrosted on ice and resuspended in 5mL LB1 (50 mM HEPES-KOH pH 7.5, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100, with 1X cOmplete Protease Inhibitor Cocktail and PMSF) and rotated vertically for 10 min at 4°C. Samples were centrifuged for 5 min at 1350 x g at 4°C, and resuspended in 5mL LB2 (10 mM Tris, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, with 1X cOmplete Protease Inhibitor Cocktail and optionally 1mM PMSF) and rotated vertically for 10 min at 4°C. Samples were centrifuged for 5 min at 1350 x g at 4°C, resuspended in 300 uL LB3 per sonicated sample, and incubated for 10 min on ice. Samples were sonicated in 1.5 mL Bioruptor Plus TPX microtubes (Diagenode, c30010010-50) on Bioruptor Plus for 10 cycles of 30sec ON/30sec OFF. Every 5 cycles, samples were lightly vortex and briefly centrifuged. Samples were diluted in additional LB3 to 1 mL, pelleted at 16,000 RCF for 10 min, and the supernatant was removed. Triton X-100 was added to 1%.
To check DNA size distribution and quantity, a 10 uL aliquot of sonicated chromatin from each sample was diluted to 100 uL in Elution Buffer (50 mM Tris, 10 mM EDTA, 1% SDS) with 0.0125 M NaCl and 0.2 mg/mL RNase A and incubated at 65C for 1 hour, followed by addition of Proteinase K to 0.2 mg/mL and an additional 1 hour of 65C incubation. DNA was purified using Zymo DNA Clean & Concentrator Kit with ChIP DNA Binding Buffer (Zymo, D5201-1-50) and size distribution and quantity was assessed by separation on a 1% agarose gel and Qubit HS DNA kit, respectively. Qubit measurements were used to normalize samples to the same DNA concentration.
Following normalization, the chromatin was divided for input (2%) and ChIP samples. A minimum of 25 ug DNA was used for histone ChIPs, and 50 ug for V5 ChIPs. 5 μg anti-H3K27ac (Active Motif, 39133) antibody or 10 μg anti-V5 (Abcam, ab9116) antibody was added per ChIP sample, and incubated overnight at 4°C. Protein G Dynabeads (ThermoFisher) were first blocked with Block solution (0.5% BSA (w/v) in 1X PBS) and then added to cleared chromatin to bind antibody-bound chromatin for a 4-6 hour incubation. Chromatin-bound Dynabeads were washed at least 6 times with chilled RIPA wash buffer (50 mM HEPES-KOH pH 7.5, 500 mM LiCl, 1 mM EDTA, 1% NP-40, 0.7% Na-Deoxycholate), followed by a wash with chilled TE + 50 mM NaCl. Chromatin was eluted for 30 min in Elution Buffer (50 mM Tris, 10 mM EDTA, 1% SDS) at 65°C with frequent vortexing. The ChIP and input samples were then incubated at 65°C overnight to reverse cross-links (12-16 hours). Samples were diluted and sequentially digested with RNase A (0.2 mg/mL) for 2 hours at 37°C followed by Proteinase K (0.2 mg/mL) for 2 hours at 55°C for 2-4 hours to digest protein. ChIP and input samples were purified by Zymo DNA Clean & Concentrator Kit with ChIP DNA Binding Buffer.
Samples were quantified by Qubit dsDNA HS assay kit, and 10-50ng of ChIP DNA was used for library preparation with end repair, A-tailing, and adaptor ligation (NEB). Following USER enzyme treatment, libraries were cleaned up with one round of single-side AMPure XP bead clean-up, then amplified to add indices using NEBNext Ultra II Q5 Master Mix and NEBNext Multiplex Oligos for Illumina kit (NEB, E7335S) with 4-6 cycles (as determined by input amounts from NEB protocol). ChIP libraries were purified by two rounds of double-sided AMPure XP bead clean-up (0.5x then 0.4x initial sample volume of beads added) to remove large fragments and deplete adaptors. Library concentration and quality within ChIP or input groups was assessed by Qubit dsDNA HS assay kit and separation on a PAGE gel, and used to pool within ChIP or input groups. KAPA qPCR was used to pool across ChIP or input groups. Pooled libraries were sequenced using Novaseq 6000 platform (2x 150bp).

tag: antibody

value: input


title: WT_R5_dTAG24h_input


title: WT_R5_untr_V5

value: V5 (Abcam ab9116)


title: WT_R5_dTAG3h_input


title: WT_R5_dTAG0h_input


title: S9CC47_R5_untr_input


title: S9CC47_R5_dTAG24h_input


title: S9CC47_R5_untr_V5


title: WT_R4_dTAG0h_input


title: S9CC47_R5_dTAG3h_input


title: WT_R4_dTAG3h_input


title: S9CC13_R4_dTAG24h_input


title: S9CC13_R4_dTAG0h_input


title: S9CC47_R5_dTAG0h_input


title: WT_R5_dTAG24h_K27ac

value: H3K27ac (Active Motif 39133)


title: S9CC13_R4_dTAG3h_input


title: WT_R5_dTAG3h_K27ac


title: S9CC47_R5_dTAG24h_K27ac


title: WT_R4_dTAG3h_K27ac


title: WT_R5_dTAG0h_K27ac


title: S9CC13_R4_dTAG24h_K27ac


title: S9CC13_R4_dTAG3h_K27ac


title: WT_R4_dTAG24h_input


title: WT_R5_V1_24h

data_processing:
Adapter sequences and low-quality bases (-Q 10) were trimmed from sequencing reads using skewer v0.2.2 and aligned to the human genome (hg38) using bowtie2 v2.4.1 with the following settings: --very-sensitive, --X 2000. Read mate pair information was corrected with samtools v1.10 fixmate, PCR duplicates were removed using samtools markdup, and mitochondrial reads and low mapping quality reads (-q 20) were removed using samtools view. bigWig files for visualization were generated using deeptools v3.5.0 bamCoverage with the following settings: -bs 10 --normalizeUsing RPGC --samFlagInclude 64 --samFlagExclude 8 --extendReads.
A custom approach based on a previous publication (Freimer et al., 2021) was used to define regions that showed reproducible peaks of accessibility across samples. Shifted bed sites were obtained from mapped and filtered ATAC bam files, and bed files for each sample were used to call peak summits using MACS2 v2.2.7.1 callpeak with the following settings: --nomodel --keep-dup all --extsize 200 --shift 100 --SPMR. Then, within each differentiation/line replicate, summits within 75bp were merged, taking the average location across summits as the location of the merged summit. Then, across each differentiation/line, summits within 150 bp were merged, again taking the average location. Only those merged summits with at least one constituent summit from three or more differentiation/line instances were carried forward. These summits were extended 250bp in either direction, and finally all such regions were merged such that there were no overlapping regions, resulting in 151,457 reproducible peak regions.
Tn5 insertion counts (from shifted bed files) were counted over reproducible peak regions using bedtools
Assembly: hg38
Supplementary files format and content: Tn5 insertion counts per reproducible peak region

extract_protocol:
ATAC-seq was performed as described previously (Corces et al., 2017). Briefly, CNCCs were incubated with BCh media containing 200U/mL DNaseI (Worthington, LS002007) for 30 min AND harvested using accutase. Viable cells were counted using Countess Automated Cell Counter (Invitrogen), 50,000 viable cells were pelleted at 500 RCF for 5 min at 4°C and resuspended in ATAC-resuspension buffer containing 0.1% NP40, 0.1% Tween20, and 0.01% Digitonin and incubated on ice for 3 minutes. Following wash-out with cold ATAC-Resuspension Buffer (RSB, 10 mM Tris-HCl pH 7.4, 10mM NaCl, 3mM MgCl2 in sterile water) containing 0.1% Tween20, cells were pelleted and resuspended in 50 μL transposition mix (25 μL 2x TD buffer, 2.5 μL transposase (100nM final), 16.5 μL PBS, 0.5 μL 1% digitonin, 0.5 μL 10% Tween20, 5 μL H2O) and incubated for 30 minutes at 37°C with shaking. The reaction was purified using the Zymo DNA Clean & Concentrator kit
Purified, transposed DNA was PCR-amplified with NEBNext master mix and primers as defined in Corces et al. Libraries were purified by two rounds of double-sided size selection with AMPure XP beads (Beckman Coulter, A63881), with the initial round of 0.5x sample volume of beads followed by a second round with 1.3x initial volume of beads. Library size distributions were confirmed by separation on a PAGE gel and staining with SYBRGold and pooled based on quantifications from Qubit dsDNA High Sensitivity Kit.


title: S9CC47_R5_dTAG3h_K27ac


title: S9CC13_R4_dTAG0h_K27ac


title: WT_R5_V1_3h


title: WT_R5_DMSO


title: WT_R4_V1_24h


title: S9CC13_R5_V1_24h


title: S9CC13_R5_DMSO


title: S9CC13_R5_V1_3h


title: WT_R4_DMSO


title: WT_R4_V1_3h


title: S9CC13_R4_V1_24h


title: S9CC13_R4_DMSO


title: S9CC47_R5_5en9


title: S9CC13_R4_V1_3h


title: S9CC47_R5_5en7


title: S9CC47_R5_dTAG0h_K27ac


title: S9CC47_R5_5en10


title: S9CC47_R5_1p6en8


title: WT_R4_dTAG0h_K27ac


title: S9CC47_R5_1p6en9


title: S9CC47_R6_5en9


title: S9CC47_R6_5en7


title: S9CC47_R5_0


title: S9CC47_R6_5en10


title: S9CC47_R6_1p6en9


title: S9CC47_R6_1p6en8


title: S9CC47_R6_0


title: S9CC13_R6_5en7


title: S9CC13_R6_5en10


title: S9CC13_R6_0


title: S9CC13_R6_1p6en8


title: S9CC13_R6_5en9


title: S9CC13_R6_1p6en9


title: S9CC13_R10_5en9


title: WT_R4_dTAG24h_K27ac


title: S9CC13_R10_5en10


title: S9CC13_R14_5en9


title: S9CC13_R14_1p6en8


title: S9CC13_R14_1p6en9


title: S9CC13_R10_1p6en9


title: S9CC13_R13_5en7


title: S9CC13_R13_5en9


title: S9CC13_R14_5en10


title: S9CC13_R14_5en7


title: S9CC13_R13_1p6en9


title: S9CC13_R13_5en10


title: S9CC13_R10_5en7


title: S9CC13_R10_1p6en8


title: WT_R8_0


title: S9CC47_R8_5en9


title: S9CC13_R13_1p6en8


title: WT_R8_5en7


title: S9CC13_R13_0


title: S9CC47_R8_5en10


title: S9CC47_R8_0


title: SK-N-BE2 shControl_rep1

city: Suzhou

first: Zimu

last: Zhang

state: Jiangsu

address: Zhongnan Street

institute: Children’s Hospital of Soochow University

data_processing:
Illumina Casava2.19 software used for basecalling.
Illumina Casava2.19 software used for basecalling.
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence, then mapped to hg38 whole genome using hisat2 v2.2.0 with parameters hisat2 [options]* -x <hisat2-idx> {-1 <m1> -2 <m2> | -U <r> | --sra-acc <SRA accession number>} [-S <hit>]
Fragments Per Kilobase of exon per Megabase of library size (FPKM) were calculated using a protocol from Trapnell,Cole et al.. In short, exons from all isoforms of a gene were merged to create one meta-transcript. The number of reads falling in the exons of this meta-transcript were counted and normalized by the size of the meta-transcript and by the size of the library.
Assembly: hg38
Supplementary files format and content: tab-delimited text files include readcount values for each Sample …

source_name: Neuroblastoma cells

extract_protocol:
Cells were harvested, and total RNA was extracted with TRIzol (Invitrogen) according to the manufacturer's protocol. Illumina TruSeq RNA Sample Prep Kit (Cat#FC-122-1001) was used with 1 ug of total RNA for the construction of sequencing libraries.
RNA libraries were prepared for sequencing using standard Illumina protocols

value: Neuroblastoma cells


title: RNAseq PE01R cell line 3

city: Nottingham

first: Nigel

middle: P

last: Mongan

address: University Park

department: Biodiscovery Institute

country: United Kingdom

institute: University of Nottingham

data_processing:
Contaminating adapters and low quality reads with phred scores <30 were removed using the Trimgalore wrapper for Cutadapt and Fastqc
The resultant quality controlled fastq files were aligned to the Ensembl annoted Human genome (GRCh38.83) using STAR
Gene expression was quantified using FeatureCounts
Assembly: GRCh38.83
Supplementary files format and content: Tab delimited text file contain gene expression in counts

source_name: PE01R cell line replicate 3

growth_protocol: Cells were maintained in vitro in RPMI culture medium (Sigma, UK) containing 10% (v/v) heat inactivated foetal bovine serum (Sigma, Poole, UK) & 2 mM L-glutamine (Sigma, UK) at 370C in 5% CO2 and humidified conditions. Cells from sub-confluent monolayers were harvested with 0.025% EDTA, washed in culture medium and counted.

extract_protocol:
RNA was extracted using Qiagen RNeasy Mini kit
Illumina

treatment_protocol: Gene expression was compared between parental PE01 and resistant PEO1R cell lines

value: Mirin resistant derivitive PE01R


title: RNAseq PE01R cell line 2

source_name: PE01R cell line replicate 2


title: WT_RSh_5en7


title: WT_RSh_0


title: RNAseq PE01R cell line 1

source_name: PE01R cell line replicate 1


title: S9CC47_R8_1p6en9


title: S9CC47_R8_5en7


title: RNAseq PE01 cell line 2

source_name: PE01 cell line replicate 2

value: Mirin sensitive parental PE01


title: RNAseq PE01 cell line 3

source_name: PE01 cell line replicate 3


title: S9CC47_R8_1p6en8


title: S9CC13_R10_0


title: Decompensated RV_7

city: Quebec City

first: Olivier

last: Boucherat

state: Quebec

address: 2325 Rue de l'Université

country: Canada

institute: Laval University

data_processing:
Basecalls was performed using bcl2fastq v1.3.0
Sequenced reads were mapped to the human genome assembly (GRCh38) with STAR v2.6.1a with the following options: STAR --readFilesCommand zcat --quantMode TranscriptomeSAM --outFilterMismatchNmax 999 --sjdbGTFfile /home/dnanexus/Homo_sapiens.GRCh38.98.gtf --genomeDir ./home/dnanexus/genome --outSAMstrandField intronMotif --sjdbScore 1 --alignIntronMin 20 --outSAMattributes NH HI AS NM MD --outSAMunmapped Within --alignSJDBoverhangMin 1 --alignIntronMax 1000000 --outSAMtype BAM SortedByCoordinate --alignSJoverhangMin 8 --alignMatesGapMax 1000000 --outFileNamePrefix /home/dnanexus/outputs/ --outFilterMismatchNoverReadLmax 0.04 --outWigStrand Unstranded --runThreadN 16
The quantification of the transcriptome-mapped reads was perfomred using RSEM v1.3.1 with the following command: rsem-calculate-expression --no-bam-output --bam --paired-end --num-threads 8
The raw counts generated from rsem were normalized using the cpm function from edgeR v3.14.0
Assembly: hg38
Supplementary files format and content: csv file for normalized genes counts for all RV samples

source_name: Heart Right Ventricle

extract_protocol:
Total RNA was extracted using Trizol (Invitrogen), according to the manufacturer’s instructions. RNA quality was checked, and RNA was quantified using a NanoDrop spectrophotometer.
RNA libraries were prepared using the TruSeq® Stranded mRNA Library

value: Right Ventricle

tag: genotype/condition

value: Decompensated


title: Decompensated RV_4


title: SK-N-BE2 shControl_rep2


title: S9CC13_R14_0


title: Decompensated RV_3


title: Decompensated RV_5


title: SK-N-BE2 shINSM2_rep1


title: SK-N-BE2 shINSM2_rep2


title: Compensated RV_9

value: Compensated


title: Compensated RV_7


title: Compensated RV_6


title: Compensated RV_5


title: RNAseq PE01 cell line 1

source_name: PE01 cell line replicate 1


title: Decompensated RV_6


title: Compensated RV_3


title: Decompensated RV_2


title: Compensated RV_2


title: Compensated RV_11


title: Decompensated RV_1


title: Compensated RV_1


title: Compensated RV_8


title: Compensated RV_10


title: Control RV_13

value: Control


title: Control RV_12


title: PSS1-WT-expressing BMC at day6 after MCSF&RANKL treatment

type: RNA

city: Tokyo

first: Nozomu

last: Kono

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

country: Japan

institute: Universtiy of Tokyo

description: Gene expression data from bone marrow cells expressing PSS1-WT at day 6 after MCSF and RANKL treatment.

hyb_protocol: Following fragmentation, 5.5 ug of cDNA were hybridized for 16 hr at 45ºC on Clarion S Mouse Array. GeneChips were washed and stained in the Genechip Fluidics Station 450.

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

data_processing: The data were analyzed with Expression ConsoleTM Software 1.4.1 using Affymetrix default analysis settings and global scaling as normalization method.

label: biotin

label_protocol: Biotinylated cDNA were prepared according to the standard Affymetrix protocol from 500 ng total RNA.

growth_protocol: Cells were cultured with α-MEM containing 10% FBS at 37 ºC, 5% CO2.

extract_protocol: Total RNA extraction was performed with High Pure RNA Isolation kit (Roche) according to the manufacturer's instructions.

treatment_protocol: Cells were treated with 50 ng/ml of MCSF and 100 ng/ml RANKL one day after seeding and infected with PSS1-expressing retroviral vector one day after treatment.

value: Bone marrow cells


title: Control RV_14


title: SJMMNORM044791_G1

city: Memphis

first: David

last: Finkelstein

state: TN

address: 332 N. Lauderdale St.

department: Computational Biology

institute: St Jude Children's Research Hospital

description: mouse brain, medulloblastoma sh2-Ift88 model

data_processing:
align using bwa sampe
count genes using Hiseq count
convert count to FPKM in R
Assembly: mm9
Supplementary files format and content: raw counts text

source_name: mouse brain, medulloblastoma model

growth_protocol: Refer to publication.

extract_protocol:
Refer to publication.
Libraries were prepared from total RNA with the TruSeq Stranded Total RNA Library Prep Kit according to the manufacturer’s instructions (Illumina, PN 20020599).

treatment_protocol: knock down of shIft88 in MB model


title: Control RV_9


title: Compensated RV_4


title: PSS1-WT-expressing BMC at day3 after MCSF&RANKL treatment

description: Gene expression data from bone marrow cells expressing PSS1-WT at day 3 after MCSF and RANKL treatment.


title: SJMMNORM044788_G1

description: mouse brain, medulloblastoma control model


title: Control RV_8


title: PSS1-LMS-expressing BMC at day6 after MCSF&RANKL treatment

description: Gene expression data from bone marrow cells expressing PSS1-LMS at day 6 after MCSF and RANKL treatment.


title: BMC at day3 after MCSF&RANKL treatment

description: Gene expression data from bone marrow cells at day 3 after MCSF and RANKL treatment.

source_name: BMC at day3 after MCSF&RANKL treatment


title: Control RV_10


title: SJMMNORM044790_G1

value: mouse brain, medulloblastoma sh2-Ift88 model


title: Control RV_7


title: SJMMNORM044786_G1

value: mouse brain, medulloblastoma control model


title: Control RV_6


title: SJMMNORM044789_G1


title: Control RV_11


title: Control RV_2


title: UI226 tumorspheres - selumetinib treated - replicate 3 [SEL_N3]

city: Winnipeg

first: Tamra

last: Werbowetski-Ogilvie

state: Manitoba

address: 611-745 Bannatyne Avenue

department: Biochemistry & Medical Genetics

institute: University of Manitoba

description: processed data file: Sel_vs_DMSO_r2_all.txt

data_processing:
Processed raw sequences with fastp to perform automatic adapter trimming, and default sequence quality filtering (>60% of bases with Q≥15, and no more than 5 'N' basecalls)
Quantified transcripts from GENCODE v35 with salmon v1.4.0 using --gcBias flag and genomic decoys in index
Performed fold change analysis using DESeq2 as follows
Read transcript quantification data with tximport library and summarized to gene level quantification
Filtered to retain genes with at least 5 reads assigned in at least two samples
Constructed DESeqDataSet object with eighteen samples (four conditions), using the model ~condition (treatment)
Calculated fold changes using the results() function, then ran lfcShrink() on the results using the apeglm method.
Genome_build: GRCh38
Supplementary_files_format_and_content: salmon transcript quantification: Quantifrication of GENCODE v35 transcripts using salmon v1.4.0
Supplementary_files_format_and_content: DESeq2 fold change results: Results from the DESeq2 lfcShrink() function comparing two conditions, including results for all genes retained after filtering

source_name: selumetinib treated tumorspheres

growth_protocol: UI226 (SHH MB as analyzed by NanoString) cells were a kind gift from Dr. Timothy Ryken (Dartmouth-Hitchcock Medical Center, New Hampshire, USA) and have been adapted to cell culture in StemPro media.

extract_protocol: RNA was extracted using the Norgen RNA extraction kit (Norgen Biotek) according to manufacturer's instructions

treatment_protocol: Tumorsphere assays were carried out in stem cell enriched conditions as described previously. Briefly, UI226 cells were plated in 24-well ultra-low attachment plates at an optimal density of 2500 cells/well and then grown for 5 days at 37°C/ 5% CO2 in either: DMSO, 2.5 µM selumetinib, 156 nM pacritinib or 2.5 µM selumetinib/156 nM pacritinib.

value: UI226 human medulloblastoma (SHH subgroup) cell line

tag: trreatment

value: selumetinib treated tumorspheres


title: SJMMNORM044787_G1


title: Control RV_4


title: BMC at day6 after MCSF treatment

description: Gene expression data from bone marrow cells at day 6 after MCSF treatment.

source_name: BMC at day6 after MCSF treatment


title: PSS1-LMS-expressing BMC at day3 after MCSF&RANKL treatment

description: Gene expression data from bone marrow cells expressing PSS1-LMS at day 3 after MCSF and RANKL treatment.


title: Control RV_1


title: BMC at day6 after MCSF&RANKL treatment

description: Gene expression data from bone marrow cells at day 6 after MCSF and RANKL treatment.

source_name: BMC at day6 after MCSF&RANKL treatment


title: UI226 tumorspheres - selumetinib treated - replicate 2 [SEL_N2]


title: Control RV_5


title: UI226 tumorspheres - selumetinib treated - replicate 1 [SEL_N1]


title: Control RV_3


title: T2_tumor tissue after chemotherapy

first: rui

last: Dong

address: 399 Wan Yuan Road

institute: Department of Pediatric Surgery, Children’s Hospital of Fudan University, and Shanghai Key Laboratory of Birth Defect

data_processing:
For droplet-base scRNA-seq, single cells were processed through the GemCode Single Cell Platform using the GemCode Gel Bead Chip and Library Kits (10x Genomics) as per the manufacturer's protocol (Zheng et al., 2017). scRNA-seq libraries were constructed using Chromium Single Cell 3.0 V2 or V3 Reagent Kit according to the manufacturer's protocol. Then, cell suspensions of each sample were run in the Chromium Controller with appropiate reagents to generate single cell gel bead-in-emulsions (GEMS) for sample and cell barcoding, with a target output of 8,000 to 12,000 cells for each sample. Amplified cDNA and final libraries were evaluated on an Agilent BioAnalyzer using a High Sensitivity DNA Kit (Agilent Technologies). The libraries were then pooled and sequenced on NovaSeq 6000 (Illumina) at a depth of approximately 400M reads per sample.
Genome_build: hg38
Supplementary_files_format_and_content: Matrix table with raw gene counts for every gene and every cell

source_name: sialoblastoma

extract_protocol: Cell suspensions of each sample were run in the Chromium Controller with appropriate reagents to generate single cell gel bead-in-emulsions for sample and cell barcoding.

value: parotid gland

value: after chemotherapy:four cycles of intermediate-risk neuroblastoma regimen

tag: age


title: UI226 tumorspheres - pacritinib + selumetinib treated - replicate 2 [PACSEL_N3]

description: processed data file: PacSel_vs_DMSO_r2_all.txt

source_name: pacritinib and selumetinib treated tumorspheres


title: T1_tumor tissue before chemotherapy

tag: tissue

value: before chemotherapy


title: UI226 tumorspheres - pacritinib + selumetinib treated - replicate 4 [PACSEL_N5]

value: pacritinib and selumetinib treated tumorspheres


title: BMC at day3 after MCSF treatment

description: Gene expression data from bone marrow cells at day 3 after MCSF treatment.

source_name: BMC at day3 after MCSF treatment


title: UI226 tumorspheres - pacritinib + selumetinib treated - replicate 3 [PACSEL_N4]


title: UI226 tumorspheres - selumetinib treated - replicate 4 [SEL_N4]


title: UI226 tumorspheres - pacritinib + selumetinib treated - replicate 1 [PACSEL_N2]


title: UI226 tumorspheres - pacritinib treated - replicate 6 [PAC_N7]

description: processed data file: Pac_vs_DMSO_r2_all.txt

source_name: pacritinib treated tumorspheres

value: pacritinib treated tumorspheres


title: RL2064_mRNAseq_DoxWD_rep4

city: Perth

first: Ryan

last: Lister

state: WA

address: 35 Stirling Highway

country: Australia

institute: The University of Western Australia

data_processing:
CASAVA v1.8.2
The WGBS sequenced reads were first trimmed using bbduk, and overlapping pairs were merged using BBMerge(Bushnell, Rood, and Singer 2017). The reads were mapped to the hg19 genome including the lambda and pUC19 sequences using BS-Seeker2 with Bowtie 2 as back-end aligner (-e 300 -X 2000)(Guo et al. 2013). The duplicated reads were removed using Sambamba markdup for paired-end reads and PALEOMIX for merged reads(Schubert et al. 2014). We then used CGmapTools to generate methylation calls(Guo et al. 2018).
Sequenced ATAC-seq reads were then trimmed with bbduk for Nextera adapters and mapped to the genome using Bowtie 2. Duplicated reads were removed and blacklisted regions filtered as with ChIP-seq, and peaks were called using MACS2 with “--nomodel -f BAM --keep-dup all” parameters. Replicates were merged using IDR, and each sample was validated visualising the insert size distribution following nucleosome periodicity
ChIP-seq reads were trimmed with bbduk(Bushnell, n.d.) and mapped to the genome using Bowtie 2(Langmead and Salzberg 2012) allowing for an insert site of 2000 bp, reads were deduplicated using Sambamba markdup function(Tarasov et al. 2015) and reads mapping to blacklisted genomic regions were filtered out using SAMtools(H. Li et al. 2009). We then used MACS2 to call peaks with the matched chromatin input sample as background for each ZF-D3A HA ChIP-seq experiment, specifying the parameters “-f BAMPE -q 0.05 --down-sample”(Feng et al. 2012). The peaks for replicate pairs were merged using IDR (https://github.com/nboley/idr)(Q. Li et al. 2011).
The RNA-seq reads were trimmed using fastp with default parameters(Chen et al. 2018), then mapped with HISAT2 (--rna-strandness RF) to an index including hg19 genome assembly and ERCC spike-in sequences(Sirén, Välimäki, and Mäkinen 2014). Then, RNA-seq read-pair counts were read into R using summarizeOverlaps function within the GenomicAlignments package and mapped to the UCSC hg19 gtf(Lawrence et al. 2013).
Genome_build: hg19 UCSC
Supplementary_files_format_and_content: Bisulfite-seq files (CGmap files) are the direct output from BSseeker2/CGmaptools (https://github.com/BSSeeker/BSseeker2), they contain chromosome position, (1) chromosome  (2) nucleotide on Watson (+) strand  (3) position  (4) context (CG/CHG/CHH)  (5) dinucleotide-context (CA/CC/CG/CT)  (6) methylation-level = #_of_C / (#_of_C + #_of_T). (7) #_of_C (methylated C, the count of reads showing C here) (8) = #_of_C + #_of_T (all Cytosines, the sum of reads showing C or T in that position).
Supplementary_files_format_and_content: NarrowPeak files are the output of MACS2 software, ZF_Counts_table_GEO.tsv.gz was computed mapping RNA-seq with HISAT2 and extracting the counts with "featureOverlaps" in R.

source_name: MCF-7 cell line - ZF-D3A-wt

growth_protocol: complete media with 1000 ng/ml doxycyline for 3 days, then just complete media for 7 days

extract_protocol:
RNAdvance Cell v2 kit. 42,252 cells were lysed using the lysis buffer from the kit and mixed with 2 µl of 1:100 diluted ERCC RNA spike-in mix 2
Illumina TruSeq Stranded mRNA kit according to the manufacturer's instructions, except one-third of all reaction volumes were used and the final amplification used 13 cycles of PCR.

tag: genotype


title: UI226 tumorspheres - pacritinib treated - replicate 5 [PAC_N5]


title: RL2060_mRNAseq_DoxMut_rep4

source_name: MCF-7 cell line - ZF-D3A-mut

growth_protocol: complete media with 1000 ng/ml doxycyline for 3 days

extract_protocol:
RNAdvance Cell v2 kit. 50,000 cells were lysed using the lysis buffer from the kit and mixed with 2 µl of 1:100 diluted ERCC RNA spike-in mix 2
Illumina TruSeq Stranded mRNA kit according to the manufacturer's instructions, except one-third of all reaction volumes were used and the final amplification used 13 cycles of PCR.


title: UI226 tumorspheres - vehicle treated - replicate 4 [DMSO_N9]

source_name: DMSO treated tumorspheres

value: vehicle control


title: UI226 tumorspheres - pacritinib treated - replicate 3 [PAC_N3]


title: UI226 tumorspheres - pacritinib treated - replicate 4 [PAC_N4]


title: RL2062_mRNAseq_DoxWD_rep2


title: RL2063_mRNAseq_DoxWD_rep3

extract_protocol:
RNAdvance Cell v2 kit. 13,734 cells were lysed using the lysis buffer from the kit and mixed with 5.49 µl of 1:200 diluted ERCC RNA spike-in mix 1
Illumina TruSeq Stranded mRNA kit according to the manufacturer's instructions, except one-third of all reaction volumes were used and the final amplification used 13 cycles of PCR.


title: RL2059_mRNAseq_DoxMut_rep3

extract_protocol:
RNAdvance Cell v2 kit. 50,000 cells were lysed using the lysis buffer from the kit and mixed with 2 µl of 1:100 diluted ERCC RNA spike-in mix 1
Illumina TruSeq Stranded mRNA kit according to the manufacturer's instructions, except one-third of all reaction volumes were used and the final amplification used 13 cycles of PCR.


title: RL2056_mRNAseq_noDoxMut_rep4

growth_protocol: complete media


title: UI226 tumorspheres - vehicle treated - replicate 2 [DMSO_N7]


title: UI226 tumorspheres - pacritinib treated - replicate 1 [PAC_N1]


title: UI226 tumorspheres - vehicle treated - replicate 1 [DMSO_N2]


title: UI226 tumorspheres - vehicle treated - replicate 3 [DMSO_N8 ]


title: RL2053_mRNAseq_noDoxMut_rep1


title: RL2055_mRNAseq_noDoxMut_rep3


title: RL2054_mRNAseq_noDoxMut_rep2


title: RL2057_mRNAseq_DoxMut_rep1


title: UI226 tumorspheres - pacritinib treated - replicate 2 [PAC_N2]


title: UI226 xenograft in NOD-SCID mouse treated with 75mg/kg selumetinib [Sel3]

data_processing:
Reads mapped independently to human (GRCh38/GENCODE v23) and mouse (GRCm38/GENCODE vM12) genome with hisat2 v2.1.0
Reads originating from the murine host removed from the dataset using XenofilteR (https://github.com/PeeperLab/XenofilteR)
Reads of human origin assigned to transcripts from GENCODE v23 using featureCounts v1.5.2
Read counts imported into R and filtered to remove non-expressed genes (retain genes with 5+ reads in at least two samples)
Differential expression computed with DESeq2 (v1.20.0) using the results() function to compare Sel vs CTRL conditions
Genome_build: GRCh38
Supplementary_files_format_and_content: featureCounts output: tab-delimited text - read counts per gene for a single sample
Supplementary_files_format_and_content: DESeq2 results output: tab-delimited text - output from the DESeq2 results() format converted to table format

growth_protocol: UI226 cells were first grown in StemPro NSC Serum-Free Medium on ultra-low attachement plates. Next, 2.5x10^5 cells were injected into the cerebellums of NOD-SCID mice

extract_protocol:
RNA was extracted from isolated human cells using a Norgen RNA extraction kit (Norgen Biotek)
Illumina TruSeq RNA v2

treatment_protocol: Xenografts were treated with either methyl cellulose 0.1% polysorbate 80 vehicle control or 75 mg/kg of selumetinib twice daily via oral gavage (drug holiday on the weekend) starting at day 14 and until endpoint.

tag: host mouse strain

value: NOD SCID

tag: injected cell line

value: 75mg/kg selumetinib

value: UI226 xenograft


title: RL2051_mRNAseq_Dox_rep3


title: UI226 xenograft in NOD-SCID mouse treated with vehicle control [CTRL3]


title: UI226 xenograft in NOD-SCID mouse treated with 75mg/kg selumetinib [Sel2]


title: RL2058_mRNAseq_DoxMut_rep2


title: RL2061_mRNAseq_DoxWD_rep1


title: UI226 xenograft in NOD-SCID mouse treated with vehicle control [CTRL2]


title: UI226 xenograft in NOD-SCID mouse treated with vehicle control [CTRL1]


title: S9CC13_R8_5e-9M

source_name: H9 hESC

value: H9 hESC

value: hESC-derived P4 CNCC


title: UI226 xenograft in NOD-SCID mouse treated with 75mg/kg selumetinib [Sel1]


title: RL2050_mRNAseq_Dox_rep2


title: RL2052_mRNAseq_Dox_rep4


title: RL2048_mRNAseq_noDox_rep4


title: RL2049_mRNAseq_Dox_rep1


title: RL2047_mRNAseq_noDox_rep3


title: THLE-2 Input for H3K27me3 2nd [ChIP-seq]

city: wuhan

first: dong

last: chen

state: Hubei

address: GaoXin 2nd Road

institute: ablife

description: processed data file: THLE2_H3K27Me3_IP_2_vs_THLE2_H3K27Me3_Input_2_peaks.txt

data_processing:
Illumina Casava1.9 software used for basecalling.
3' adapter (end1:AGATCGGAAGAGC;end2:AGATCGGAAGAGC) trimmed with cutadapt.
Reads were mapped using bowtie2 -N 1. Unique mapped reads were remained.
MACS version 1.4.2 was used to peakcalling with default parameters.
Genome_build: GRCh38
Supplementary_files_format_and_content: *_peaks.txt: Tab-delimited text files include peak information for each group.

source_name: cell line

extract_protocol:
The cells lysates was sonicated to generate DNA fragments of 200-500 bp, centrifuged for 10 min at 12,000 g, and the supernatant was directly used for immunoprecipitation. Place the tube containing the Pierce™ ChIP-grade Protein A/G Magnetic Beads on the magnetic, then, discard the supernatant. Wash the beads for three times with 1ml 0.1M sodium phosphate Buffer. Resuspend the beads with 200ul 0.1M sodium phosphate Buffer. Add 10 ug target antibody (CST, 5646s) and Rabbit IgG (CST, 2729s) antibody to the tube containing beads. Incubate mixture on a rotating wheel 2h at 4°C. Place the tube containing the beads on the magnetic stand for 1 minute. Then, discard the supernatant. Resuspend beads with the cells lysates. Rotate the sample for overnight at 4 °C. Place on the magnetic, then, discard the supernatant. The beads were sequentially washed with LiCl IP Wash Buffer (100 mM Tris (PH 7.5), 500 mM LiCl, 1% NP-40 and 0.5 deoxycholate) for five times, TE buffer (10Mm Tris (PH 8.0), 1mM EDTA (PH 8.0)) for one time, respectively. Resuspend sample with 100ul Elution Buffer (100 mM NaHCO3, 1% SDS) and reverse cross-linked by overnight incubation at 65 ℃. After sequential RNase A and proteinase K treatment, DNA fragments were purified by phenol extraction and ethanol precipitation. Resuspend the RNA pellet in10ul water.
To generate libraries with the ThruPLEX® DNA-seq Kit (R400427, Rubicon Genomics) according the manufacturer’s procedure. Briefly, purified DNA fragments were end-repaired, adenylated, ligated to adaptors and PCR amplification. Used the ThruPLEX® DNA-seq Kit. For high-throughput sequencing, the libraries were prepared following the manufacturer's instructions and applied to Illumina NextSeq 500 system for 100 nt pair-end sequencing by ABlife, Inc (Wuhan, China).

treatment_protocol: Removing the growth media and then rinse the cells thrice with cold 1XPBS and remove the supernatant. Add formaldehyde to a final concentration of 1%, mix gently, and incubate at room temperature for 10 minutes.

value: normal liver epithelial cells

tag: chip antibody


title: RL1950_WGBS_DoxMut_rep1

extract_protocol:
Qiagen DNeasy Blood & Tissue Kit
500 ng of genomic DNA was spiked with 0.5% (w/w) of unmethylated lambda phage DNA (Promega) for calculation of the bisulfite non-conversion rate, and sheared with a Covaris S2 sonicator to an average length of 300 bp. The sheared DNA was end-repaired, A-tailed and ligated to methylated adapters (NEXTflex Bisulfite-Seq Barcodes, PerkinElmer) using the NxSeq AmpFREE Low DNA Library Kit (Lucigen). Adapter ligated libraries were subject bisulfite conversion using the EZ DNA Methylation-Direct Kit (Zymo) following the manufacturer’s instructions and subjected to 6 cycles of PCR amplification using KAPA HiFi Uracil+ DNA polymerase (KAPA Biosystems).


title: THLE-2 Input for H3K27me3 1st [ChIP-seq]

description: processed data file: THLE2_H3K27Me3_IP_1_vs_THLE2_H3K27Me3_Input_1_peaks.txt


title: THLE-2 H3K27me3 1st [ChIP-seq]

description:
Pulldown DNA by chromatin immunoprecipitation.
processed data file: THLE2_H3K27Me3_IP_1_vs_THLE2_H3K27Me3_Input_1_peaks.txt


title: Bb

city: STRASBOURG

first: Nicolas

last: MATT

address: 15 rue René Descarte

department: IBMC

country: France

institute: CNRS

description: B. bassiana induced flies

data_processing:
Instrument version: “Hi-Seq 2000”
Sequencing kit: TruSeq(TM) SBS v5
Number of sequencing cycles: 1 x 50 + 7 (index*)
HiSeq Control Soft. v. 1.4.8, RTA 1.12.4.2, CASAVA 1.8.2 for the Base calling Pipeline
Genome_build: dm5
Supplementary_files_format_and_content: 040112_align22_Dm_mature-miRNA_CtrlMlBb.tab
Supplementary_files_format_and_content: Counts of 22 na length sequences alligning to knowned drosophila mature miRNA

organism: Drosophila melanogaster

source_name: Bb

growth_protocol: Flies were raised on standard cornmeal-yeast-agar medium at 25°C with 60% humidity.

extract_protocol:
RNA was extracted from 20 whole flies and treated with DNAse, using RNA Spin kit (Macherey Nagel).18-25nt RNAs were cut from a 15% acrylamide gel after electrophoresis.
RNA libraries were prepared for sequencing using NEBNext Small RNA Sample Prep Set1

treatment_protocol: Wild-type male flies were infected by pricking with M. luteus or natural infection with B. bassiana and killed respectively 12h and 24h after infection.

value: wild type

tag: agent

value: B. bassiana


title: RL1948_WGBS_DoxWD_rep2


title: RL1949_WGBS_noDoxMut_rep1


title: THLE-2 H3K27me3 2nd [ChIP-seq]

description:
Pulldown DNA by chromatin immunoprecipitation.
processed data file: THLE2_H3K27Me3_IP_2_vs_THLE2_H3K27Me3_Input_2_peaks.txt

tag: cell type


title: THLE-2 Input for JARID2 1st [ChIP-seq]

description: processed data file: THLE2_Jarid2_IP_1_vs_THLE2_Jarid2_Input_1_peaks.txt


title: THLE-2 Input for JARID2 2nd [ChIP-seq]

description: processed data file: THLE2_Jarid2_IP_2_vs_THLE2_Jarid2_Input_2_peaks.txt


title: THLE-2 JARID2 2nd [ChIP-seq]

description:
Pulldown DNA by chromatin immunoprecipitation.
processed data file: THLE2_Jarid2_IP_2_vs_THLE2_Jarid2_Input_2_peaks.txt


title: Control

description: Non induced flies

source_name: Control


title: Ml

description: M. luteus induced flies

source_name: Ml

value: M. luteus


title: RL1947_WGBS_DoxWD_rep1


title: cydAB 3

city: Nashville

first: Maria

last: Hadjifrangiskou

address: 1211 Medical Center Drive

department: Pathology, Microbiology, and Immunology

institute: Vanderbilt University Medical Center

description: urothelial cells intracellularly infected with UPEC

hyb_protocol: 100 ng purified RNA (20 ng/µL) was hybridized for 20 hours and analyzed using the nCounter Human Metabolic Pathways Panel (NanoString Technologies) according to manufacturer’s protocols.

scan_protocol: Arrays were scanned according to manufacturer's protocols.

data_processing: Raw data were normalized and subjected to background thresholding using default settings on nSolver v4.0

source_name: 5637 human transitional bladder epithelial cells (ATCC HTB-9)

growth_protocol: HTB-9 urothelial cells were propagated and infected in RPMI (Gibco) supplemented with 10% fetal bovine serum (Gibco).

extract_protocol: RNA was extracted was intracellularly infected urothelial cells using TRIzol Reagent (Invitrogen) according to manufacturer’s protocols. 20 µg nuclease free glycogen (Sigma) was added to facilitate RNA precipitation.

treatment_protocol: Cells were mock infected or infected either with UPEC isolate UTI89 or an isogenic mutant lacking cytochrome bd (∆cydAB). Bacteria were added to urothelial cell monolayers at a multiplicity of infection (MOI) of 5-10, centrifuged at 600 x g for 5 minutes to facilitate and synchronize attachment, and incubated for two hours at 37˚C in 5% CO2. Monolayers were washed thoroughly with PBS, and fresh RPMI containing 100 µg/mL gentamicin (Gibco) was added to each well to kill extracellular bacteria. Monolayers were incubated another two hours and washed with PBS prior to RNA extraction.

value: HTB-9 urothelial cell

tag: infecting strain


title: THLE-2 JARID2 1st [ChIP-seq]

description:
Pulldown DNA by chromatin immunoprecipitation.
processed data file: THLE2_Jarid2_IP_1_vs_THLE2_Jarid2_Input_1_peaks.txt

tag: cell line


title: cydAB 1


title: cydAB 2


title: THLE-2 Input for EZH2 2nd [ChIP-seq]

description: processed data file: THLE2_EZH2_IP_2_vs_THLE2_EZH2_Input_2_peaks.txt


title: RL2046_mRNAseq_noDox_rep2


title: UTI89 4


title: RL2045_mRNAseq_noDox_rep1


title: ShMETTL3-input MeRIP

city: Jinan

first: Chunyan

last: Ji

address: Wenhua West Road

institute: Qilu Hospital

data_processing:
QC check
Read mapping (hisat2)
Peakcalling (exome peak)
RNA methylaytion differenetial analysis
Assembly: mm10
Supplementary files format and content: bigWig

source_name: Jurkat

extract_protocol:
The total RNA was extracted by an EASYspin Plus kit (Yishan, China) and dissolved in 40 µL of RNase-free water. Then, the cells were divided into two groups, one group for input and the other group was increased to 1 mL by the addition of buffer containing an RNase inhibitor, ribonucleoside vanadyl complexes, an m6A-specific antibody (Sigma–Aldrich) or rabbit IgG (Sigma Aldrich) and incubated for 2 h at 4 °C. Next, the prewashed beads A were added and incubated for 2 h. Elution buffer containing an anti-m6A antibody (Synaptic Systems) was added to the mixture and incubated for 1 hour with continuous shaking at 4 °C. The methylated mRNA was precipitated with 5 mg of glycogen and one-tenth volumes of 3 M sodium acetate in a 2.5 volume of 100% ethanol at -80 °C overnight.
For each sample, 1 μg of the total RNA was used for the RNA-seq library preparation by a VAHTS Stranded mRNA-sequence Library Prep Kit (Vazyme, China). Polyadenylated mRNAs were purified, fragmented and then converted into double-stranded cDNA. After end repair and A tailing, the DNA was ligated to VAHTS RNA Adapters (Vazyme, China). Purified ligation products corresponding to 200-500 bps were digested with heat-labile UDG, and the single strand cDNA was amplified, purified, quantified and stored at -80 °C before sequencing

treatment_protocol: Jurkat cells were transfected with shMettl3 virus or shControl virus

value: Jurkat

value: T-cell acute lymphoblastic leukemia

value: WT

tag: treatment

value: shMettl3 virus


title: MMC, replicate 1, scRNAseq

city: Hamburg

first: Yu

last: Zhao

address: Falkenried 94

department: Institute of Medical Systems Biology

country: Germany

institute: University Medical Center Hamburg-Eppendorf

description: 10x Genomics

library_source: transcriptomic single cell

data_processing:
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)
Assembly: mm10
Supplementary files format and content: Tab-separated values files and matrix files

source_name: Fetal brain

extract_protocol:
Embryonic day 18.5 old fetuses were isolated from the maternal uterus and sacrificed by decapitation. Fetal brains were then disrupted mechanically and washed through a 40µm cell strainer with PBS. Unspecific FcγRII/III binding was blocked by incubating cells with normal rat serum and TruStain fcX (anti-mouse CD16/32, BioLegend, Cat. No. 101320). Afterwards, cells were stained with Fixable viability dye eFluor506TM, anti-CD45.1 (1:400, BioLegend, Cat. No. 110705), anti-CD45.2 (1:100, BioLegend, Cat. No. 109823), anti-H-2Db/b (1:20, BioLegend, Cat. No. 111507), anti-H-2Dd/d (1:200, BioLegend, Cat. No. 114712). Cells were sorted using the FACSAria III flow sorter (BD Life Sciences) with the Diva Software.
Single cells were encapsulated in droplets using GemCode Technology and processed by using Chromium Single Cell 5’ Reagent Kits (v2, 10X Genomics) following manufacturer’s specifications. Briefly, transcriptome from each cell was captured in single droplet, and hybridized to gel beads with unique cell barcode and unique molecular identifier (UMI). Subsequently, barcoded cDNA was transcribed in droplets and then released for library construction. The constructed libraries were sequenced by HiSeq 4000 (Illumina) in paired-end to reach approximately 50.000 reads per cell.

value: Maternal immune cells

value: Fetal brain

tag: strain

value: Embryonic day 18.5


title: ShMETTL3 MeRIP


title: RL1945_WGBS_Dox_rep1


title: UTI89 3


title: THLE-2 Input for EZH2 1st [ChIP-seq]

description: processed data file: THLE2_EZH2_IP_1_vs_THLE2_EZH2_Input_1_peaks.txt


title: THLE-2 EZH2 2nd [ChIP-seq]

description:
Pulldown DNA by chromatin immunoprecipitation.
processed data file: THLE2_EZH2_IP_2_vs_THLE2_EZH2_Input_2_peaks.txt


title: UTI89 2


title: RL1944_WGBS_noDox_rep2


title: RL1943_WGBS_noDox_rep1


title: RL1926_ATACseq_DoxMut_rep2

extract_protocol:
50,000 FACS purified cells were resuspended in 50 µl cold ATAC resuspension buffer 1 (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% (v/v) NP40, 0.1% (v/v) Tween-20, 0.01% (v/v) Digitonin) and incubated on ice for 3 minutes to lyse cells. The lysis reaction was stopped by adding 1 ml of cold ATAC resuspension buffer 2 (10 mM Tris-HCl pH 7.5, 10 mM NaCl, 3 mM MgCl2, 0.1% (v/v) Tween-20) and inverting the tube for 3 times to mix. The nuclei were pelleted by centrifugation at 500xg for 10 minutes at 4℃ in a fixed angle centrifuge.
The supernatant was removed and the nuclei were resuspended in transposition mix containing 25 µl of 2x TD buffer (20 mM Tris-HCl pH 7.5, 10 mM MgCl2, 20% (v/v) Dimethyl Formamide), 2.5 µl of 100 nM Tn5, 16.5 µl PBS, 0.5 µl of 1% (v/v) digitonin, 0.5 µl of 10% (v/v) Tween-20, and 5 µl H2O. The transposition reaction was incubated at 55°C for 30 minutes in a thermomixer with 1000 RPM mixing. Transposed DNA was cleaned up using the Qiagen MinElute PCR Purification kit following manufacturer’s instructions. 20 µl of eluted DNA was subject to 8-10 cycles of PCR amplifications using NEBNext 2x MasterMix.


title: ShControl MeRIP

value: shControl virus


title: THLE-2 EZH2 1st [ChIP-seq]

description:
Pulldown DNA by chromatin immunoprecipitation.
processed data file: THLE2_EZH2_IP_1_vs_THLE2_EZH2_Input_1_peaks.txt


title: RL1946_WGBS_Dox_rep2


title: HepG2 Input for JARID2 2nd [ChIP-seq]

description: processed data file: HepG2_Jarid2_IP_2_vs_HepG2_Jarid2_Input_2_peaks.txt

value: liver carcinoma cells


title: UTI89 1


title: RL1924_ATACseq_Dox_rep2


title: mock 4


title: BMDM Elp3 KO IL4 treated rep1

city: Liège

first: Dawei

last: Chen

address: Avenue de l'Hôpital,11

department: GIGA-Stem cells

country: Belgium

institute: University of Liège

data_processing:
Basecalls performed using bcl2fastq version2.20
RNA-seq reads were aligned to the GRCm38 genome Ensembl assembly (release 97) using STAR version 2.7.1a
Raw gene counts were generated by adding --quantMode GeneCounts to the STAR command
Genome_build: GRCm38
Supplementary_files_format_and_content: Matrix table with raw gene counts for every gene and every sample

source_name: bone marrow derived macrophages

growth_protocol: DMEM medilum with 20% L929 cultured medium

extract_protocol:
Bone marrow was extracted from femur and tibia, after lysis of erythrocytes
Illumina Truseq stranded mRNA library preparation from 1 µg Total RNA.

treatment_protocol: IL-4, 2 hours, 10ng/ml

value: bone marrow derived macrophages

value: Elp3 KO


title: mock 3


title: RL1923_ATACseq_noDox_rep2


title: HepG2 Input for JARID2 1st [ChIP-seq]

description: processed data file: HepG2_Jarid2_IP_1_vs_HepG2_Jarid2_Input_1_peaks.txt


title: HepG2 JARID2 2nd [ChIP-seq]

description:
Pulldown DNA by chromatin immunoprecipitation.
processed data file: HepG2_Jarid2_IP_2_vs_HepG2_Jarid2_Input_2_peaks.txt


title: BMDM Elp3 KO IL4-Untreated rep3


title: mock 2


title: ShControl-input MeRIP


title: HepG2 JARID2 1st [ChIP-seq]

description:
Pulldown DNA by chromatin immunoprecipitation.
processed data file: HepG2_Jarid2_IP_1_vs_HepG2_Jarid2_Input_1_peaks.txt

extract_protocol:
The cells lysates was sonicated to generate DNA fragments of 200-500 bp, centrifuged for 10 min at 12,000 g, and the supernatant was directly used for immunoprecipitation. Place the tube containing the Pierce™ ChIP-grade Protein A/G Magnetic Beads on the magnetic, then, discard the supernatant. Wash the beads for three times with 1ml 0.1M sodium phosphate Buffer. Resuspend the beads with 200ul 0.1M sodium phosphate Buffer. Add 10 ug JARID2 antibody (Novus Biological, NB100-2214) and Rabbit IgG (CST, 2729s) antibody to the tube containing beads. Incubate mixture on a rotating wheel 2h at 4°C. Place the tube containing the beads on the magnetic stand for 1 minute. Then, discard the supernatant. Resuspend beads with the cells lysates. Rotate the sample for overnight at 4 °C. Place on the magnetic, then, discard the supernatant. The beads were sequentially washed with LiCl IP Wash Buffer (100 mM Tris (PH 7.5), 500 mM LiCl, 1% NP-40 and 0.5 deoxycholate) for five times, TE buffer (10Mm Tris (PH 8.0), 1mM EDTA (PH 8.0)) for one time, respectively. Resuspend sample with 100ul Elution Buffer (100 mM NaHCO3, 1% SDS) and reverse cross-linked by overnight incubation at 65 ℃. After sequential RNase A and proteinase K treatment, DNA fragments were purified by phenol extraction and ethanol precipitation. Resuspend the RNA pellet in10ul water.
To generate libraries with the ThruPLEX® DNA-seq Kit (R400427, Rubicon Genomics) according the manufacturer’s procedure. Briefly, purified DNA fragments were end-repaired, adenylated, ligated to adaptors and PCR amplification. Used the ThruPLEX® DNA-seq Kit. For high-throughput sequencing, the libraries were prepared following the manufacturer's instructions and applied to Illumina NextSeq 500 system for 100 nt pair-end sequencing by ABlife, Inc (Wuhan, China).


title: BMDM Elp3 KO IL4-Untreated rep2


title: RL1922_ATACseq_DoxMut_rep1


title: RL1921_ATACseq_DoxWD_rep1


title: BMDM Elp3 KO IL4 treated rep3


title: BMDM Elp3 KO IL4 treated rep2


title: BMDM Elp3 KO IL4-Untreated rep1


title: THLE-2 IgG for JARID2 2nd [CLIP-seq]

description:
Pulldown RNA by immunoprecipitation.
processed data file: THLE2_Jarid2_IP_2_peak_info.txt

data_processing:
Library strategy: CLIP-seq
Illumina Casava1.9 software used for basecalling.
3' adapter (end1:TGGAATTCTCGGGTGCCAAGG;end2:GATCGTCGGACTGTAGAACT) trimmed with fastx_clipper.
Reads were mapped using tophat2 -N 4. Unique mapped reads were remained. While several mapped reads have the same start position and end position, only one is considered.
Reads with overlap region on the locus of genome were clustered. The read number (N), read length and observed max depth were then counted for each cluster. For each gene, a same number of reads with coincident lengths were generated using computational simulation. The outputting reads were further randomly placed to same genes to generate max peak height from overlapping reads. The whole process was repeated for 500 times. All the observed peaks with heights higher than those of random max peaks (p value ＜ 0.01) were selected, while the EZH2 and JARID2 peaks that have an overlap with control peaks were removed.
Genome_build: GRCh38
Supplementary_files_format_and_content: *_peak_info.txt: Tab-delimited text files include peak information for each group.

extract_protocol:
For immunoprecipitation, the supernatant was incubated overnight at 4 °C with 15ug antibody or control IgG antibody.
Carry the recovered RNA through a standard cDNA library preparation protocol originally described for cloning of small regulatory RNAs. For high-throughput sequencing, the libraries were prepared following the manufacturer's instructions and applied to Illumina NextSeq 500 system for 100 nt pair-end sequencing by ABlife, Inc (Wuhan, China).

treatment_protocol: Cells were cross-linked on ice with UV irradiation type C (254 nm) at 400 mJ/cm2 in the presence of cold PBS (4 ml per 15-cm dish).

value: Normal liver epithelial cells

value: IgG


title: HC11, EpPT1 3

city: Kaifeng

first: Haigang

last: wu

state: Henan province

address: dongjing Road

department: School of Life Sciences

institute: Henan University

data_processing:
Illumina Casava1.7 software used for basecalling
Sequenced reads were trimed for adaptor sequence, and masked for low-complexity or low quality sequence, then mapped to the mm8 whole genome using bowtie v0.12.2 with parameters set as defaults.
Reads for kilobased of exon per megabase of library size (RPKM) were calculated using a protocol from Chepelev et al., Nucleic Acids Research, 2009.
Assembly: mm8
Supplementary files format and content: counts

source_name: HC11, cells

growth_protocol: Mouse normal breast  epithelial cells HC11, were purchased from the American Type Culture Collection (ATCC, USA). Cells were cultured in complete medium, DMEM (Gibco) containing 10% Fetal Bovine Serum (FBS, Cat.10270-106, Gibco), 100 unit penicillin-streptomycin (Cat. 15140122, Gibco). The culturing environment was set as 5% CO2 at 37 ℃.

extract_protocol:
Cell samples were lysed and harvested using Trizol reagent. THe purification of RNA was performed using protocol provided by extracting kits (Total RNA Extraction Kit, Cat. R1200, Solarbio life science, China).
RNA library were prepared for sequencnig using standard illumina protocols

treatment_protocol: HC11, cells in 12-well dish were exposed to 660 nm laser (10 mW, 0.5 J/cm2 and 1 J/cm2). Then, total RNA of HC11, cells were extracted with Total RNA Extraction Kit (Solarbio, Shanghai, China).

value: HC11, cells

value: mammary epithelial cells


title: RL1920_ATACseq_Dox_rep1


title: THLE-2 JARID2 2nd [CLIP-seq]


title: RL1925_ATACseq_DoxWD_rep2


title: HC11, EpPT2 2


title: BMDM WT IL4 treated rep3


title: HC11, EpPT2 1


title: RL1919_ATACseq_noDox_rep1


title: HC11, EpPT2 3


title: BMDM WT IL4 treated rep1


title: HC11, EpPT1 1


title: BMDM WT IL4 treated rep2


title: mock 1


title: THLE-2 JARID2 1st [CLIP-seq]

description:
Pulldown RNA by immunoprecipitation.
processed data file: THLE2_Jarid2_IP_1_peak_info.txt


title: HC11, EpPT1 2


title: THLE-2 IgG for EZH2 2nd [CLIP-seq]

description:
Pulldown RNA by immunoprecipitation.
processed data file: THLE2_EZH2_IP_2_peak_info.txt


title: RL1901_input_Dox

extract_protocol:
Cells were washed two times with 10 ml of PBS and crosslinked for 5 minutes in 50 mM HEPES-KOH, pH 7.5, 100 mM NaCl, 1mM EDTA, 1% formaldehyde. The crosslinking reaction was quenched by the addition of glycine to a final concentration of 125 mM and washed twice with phosphate buffered saline (PBS). All subsequent solutions were supplemented with a protease inhibitor cocktail (Sigma Cat. # P8340). Cells were scraped off the plates with a rubber policeman in 10 ml of PBS and centrifuged at 3,000 rpm for 5 minutes in a swinging bucket rotor. The cell pellets were resuspended in 10 ml of 50 mM HEPES-KOH, pH 7.9, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100, incubated on ice for 10 minutes and centrifuged at 3,000 rpm for 10 minutes in a swinging bucket rotor. Cell pellets were washed twice by gently adding 10 mM Tris-HCl, pH 8.1, 200 mM NaCl, 1 mM EDTA to the cell pellets, trying not to disturb the pellets, and centrifuged at 3,000 rpm for 5 min. Finally the cell pellets were resuspended in 0.1% SDS, 1 mM EDTA and transferred to a Covaris TC12x12 tube. The chromatin was sheared using a Covaris S2 sonicator with the following settings: time 12 min, duty cycle 5%, intensity 4, cycles per burst 200, temperature 4˚C, power mode frequency sweeping. Triton X-100 and NaCl were added to a final concentration of 1% and 150 mM respectively. The sheared chromatin was centrifuged at maximum speed in a microfuge for 15 minutes at 4˚C and the supernatant was transferred to a new tube.
Genomic DNA was fragmented with a Covaris S2 sonicator to a mean length of 200 bp, then end-repaired, A-tailed, ligated to methylated Illumina TruSeq adapters, bisulfite converted and subjected to 7 cycles of PCR amplification with KAPA HiFi Uracil+ DNA polymerase (KAPA Biosystems).


title: BMDM WT IL4-Untreated rep3


title: THLE-2 IgG for JARID2 1st [CLIP-seq]


title: HC11, Ctrl 2

value: untreated


title: THLE-2 IgG for EZH2 1st [CLIP-seq]

description:
Pulldown RNA by immunoprecipitation.
processed data file: THLE2_EZH2_IP_1_peak_info.txt


title: HC11, Ctrl 3


title: RL1900_ChIPseq_DoxMut_HAflag_rep2


title: THLE-2 EZH2 1st [CLIP-seq]


title: BMDM WT IL4-Untreated rep1


title: RL1902_input_DoxMut


title: RL1897_ChIPseq_Dox_HAflag_rep1


title: HepG2 IgG for JARID2 2nd [CLIP-seq]

description:
Pulldown RNA by immunoprecipitation.
processed data file: HepG2_Jarid2_IP_2_peak_info.txt

value: Liver carcinoma cells


title: WT_H2O_1

city: Berlin

first: Benedikt

last: Obermayer

address: Chariteplatz 1

department: Core Unit Bioinformatics

institute: Berlin Institute of Health

data_processing:
Sequencing libraries were processed with CellRanger (v3.1.0) against the mouse genome (mm10) augmented by the APP and PS1 transgenes
background removal with CellBender
snRNA-seq data analysis was performed in R (v3.6.3) with Seurat (v3.2.1)
Cells with at least 250 but less than 6000 genes and less than 10% mitochondrial RNA content were combined from each library, and clustering and UMAP embedding was computed based on log normalized gene counts, with total RNA content regressed out during scaling and using 20 PCA components
Assembly: mm10 + APP/PS1 transgenes
Supplementary files format and content: raw cellranger count matrix (h5 format)
Supplementary files format and content: final seurat object

source_name: Cerebrum

extract_protocol:
Mouse hemispheres were harvested from male mice at the age of 180 days and immediately snap frozen in liquid nitrogen and stored at -80°C until further processing for nuclei isolation. Nuclei were isolated from a single mouse hemisphere in 2 ml of pre-chilled EZ PREP lysis buffer (NUC-101, Sigma) using a glass Dounce tissue grinder (D8938, Sigma) (25 strokes with pastel A and 25 strokes with pastel B) followed by incubation for 5 minutes on ice with additional 6 ml of EZ PREP buffer. During incubation, 1 µM DAPI was added to the homogenate and subsequently filtered through a 35 µm strainer. Intact nuclei were sorted with a BD FACSAriaIII with a 70 µm configuration into 1.5 ml-Eppendorf tubes with 40 µl of 4 % BSA in PBS and RiboLock RNase Inhibitor (25 U/µl, EO0381, ThermoFisher). A FSC/SSC based gate was used to exclude debris followed by exclusion of damaged nuclei in a DAPI-A/DAPI-H (see Supplementary Fig. 5a). 150,000 events were sorted for each sample. The concentration of sorted nuclei was determined based on brightfield images and DAPI fluorescence using a Neubauer counting chamber and a Leica DMi8 microscope.
Single nuclei libraries were generated according to the Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1 User Guide (CG000204) by 10x Genomics. Briefly, a droplet emulsion was generated in a microfluidic chip followed by barcoded cDNA generation inside these droplets. Purified and amplified cDNA was then subjected to library preparation and sequenced on a NovaSeq 6000 instrument (Illumina) to a median depth of 45-90k reads per cell.

treatment_protocol: APPPS1+/− mice and littermate wild type control (WT) mice were treated with 3 mM spermidine dissolved in their drinking water (changed twice a week) from an age of 30 days until an age of 180 days. Control mice received only water (H2O). Prior to each exchange of the drinking bottles, the weight of the bottles was determined and used to calculate the average volume consumed per animal per day. Animals were kept in individually ventilated cages with a 12 h light cycle with food and water ad libitum.

value: Cerebrum

value: 180 days

tag: Sex

value: male


title: EC, brDura, naive, 2

city: Goettingen

first: Gabriela

last: Salinas

state: Lower-Saxony

address: Kreuzbergring 57

department: Department of Pathology

institute: Universitaetsmedizin Goettingen

description: CD31+ endothelial cells sorted from naïve brain dura

data_processing:
Sequence images were transformed with Illumina software BaseCaller to BCL files, which was demultiplexed to fastq files with bcl2fastq v2.20
STAR version 2.5 was used to align the reads to the rn6 genome using 2 mismatches within 50 bases
featureCounts version 1.5.0-p1 was used to quantify reads for genes in rn6 version 87
Assembly: rn6 version 87
Supplementary files format and content: Tab-delimited gene counts

organism: Rattus norvegicus

source_name: Brain dura

extract_protocol:
CD31+ endothelial cells were isolated from tissues by digestion with 0.4 U ml-1 Liberase TM and 60 U ml-1 of DNase1 subsequently FACS sorted. Total RNA was purified from sorted cells using the TRIzol protocol (Invitrogen)
Library preparation was performed using the TruSeq RNA Sample Preparation Kit (Illumina, Cat. N°RS–122–2002) starting from 100-500ng of total RNA

value: Brain dura

value: Lewis

value: wt

value: CD31+ CD45- CD11b- endothelial cells (sorted)

tag: time point

value: naïve animals


title: RL1899_ChIPseq_Dox_HAflag_rep2


title: BMDM WT IL4-Untreated rep2

value: WT


title: HC11, Ctrl 1


title: HepG2 JARID2 2nd [CLIP-seq]

tag: antibody