title: Mammary tumor from N6099 (control) mouse [N-15]


title: H60, >120 μm, replicate 1, scRNAseq

status: Public on Apr 11 2024

type: SRA

city: Celbridge

first: Lais

last: Barbosa Latorraca

state: Kildare

address: UCD Lyons Farm

department: Agriculture and Food Science

country: Ireland

institute: University College Dublin

library_source: transcriptomic single cell

data_processing:
Trim Galore (version 0.6.6) was used to trim raw sequence reads by removing adapter contamination and poor-quality reads with less than 20 Phred score. Only reads with a minimum 20 bp sequence length after trimming were retained for further processing. The data was mapped to the ARS-UCD 1.2 bovine genome using Hista2. Raw counts were quantified using SeqMonk software (version 11.0.10; Babraham Institute; https://www.bioinformatics.babraham.ac.uk/projects/seqmonk/).
Assembly: ARS-UCD 1.2
Supplementary files format and content: Tab-Delimited Text file containing raw read counts for each sample.

molecule: polyA RNA

organism: Bos taurus

source_name: Oocyte

extract_protocol:
PCR tubes containing single oocyte samples snap-frozen in PBS + RLT-Plus Buffer at the collection day were transported in dry ice to the Babraham Institute, UK. Oocyte lysates were thawed and transferred to a 96-well plate and incubated with Dynabeads (MyOne Streptavidin C1, Life Technologies) annealed to Smart-seq2 oligo-dTs to capture polyadenylated mRNA. The remaining lysate containing the DNA was transferred to a new 96 well-plate for a subsequent bisulphite conversion analysis. The beads containing the mRNA were diluted in reverse transcription master mix using SuperScript™ II Reverse Transcriptase (Invitrogen) and reverse transcription was performed to produce cDNA.
cDNA was amplified using KAPA HiFi HotStart ReadyMix (Roche), purified with Ampure XP beads with a 1:0.9 ratio, and eluted into 20 μL of water. Amplified cDNA was quantified using a High Sensitivity DNA Assay chip (Agilent Technologies), and libraries were prepared using the Nextera XT Kit (Illumina) with ~300 pg of cDNA in two different replicates. A total of 179 oocytes were sequenced on the NextSeq500 HighOutput 150 bp Single End (replicate 1) and 75 bp Single End (replicate 2) with a sequence depth of ~2 million reads.

tag: cell type

value: Oocyte

tag: experimental group

tag: diameter (um)


title: mEpiSC-11_control

city: Beijing

first: Jiansen

last: Lu

address: No. 5 Yiheyuan Road, Haidian District

department: Biomedical Pioneering Innovation Center (BIOPIC)

country: China

institute: Peking University

description: Control group of CLEVER-seq

library_source: genomic

data_processing:
All the de-multiplexed fastq reads were stripped to remove the low-quality bases and artificial sequences, including MALBAC primer and illumina adaptor sequences using trim galore (version: 0.3.3). The remaining cleaned reads were then aligned to the mm9 mouse and hg19 human reference genome using the Bismark tool (version: 0.23.0)  with the default parameters (Krueger and Andrews, 2011). Duplicated reads were removed using samtools (version: 1.9) (Li et al., 2009).
bigwig files were generated using the 'bamcoverage' module of deeptools (version 3.5.5).
Assembly: mm9 mouse
Supplementary files format and content: bigwig files contains the genome coverage

molecule: genomic DNA

organism: Mus musculus

extract_protocol:
Single mouse/human embryonic stem cells, gametes and early embryos were picked into the lysis buffer (20 mMTris, 2 mM EDTA, 20 mMKCl, and 0.3% TritonX-100) using a mouth pipette. Genomic DNAs were released after treatment with proteinase (QIAGEN) at 50°C for 3 h. To label the 5fC adequately, 150 mM malononitrile (J&K) was added directly to the single cell lysate, which was allowed to react at 37°C for 20 h with continuously shaking in a thermomixer (Eppendorf, 850 r.p.m.). For the control group, same volume of H2O was used instead of malononitrile. To obtain the whole genome 5fC profiles from the single cells, MALBAC technique was applied to amplify the malononitrile-labeled genomic DNAs with minor modifications(Zong et al., 2012). Briefly, 11 rounds of quasi-linear pre-amplification and 15 cycles of exponential amplification were performed to obtain sufficient DNA for library construction.
Sequencing libraries were constructed under the instruction of NEBNext Ultra DNA Library Prep Kit for Illumina (NEB), and the final libraries were amplified using the DeepVent DNA polymerase (3’-5’ exo-, NEB).

value: mouse epiblast stem cell

tag: treatment

value: Control group using H2O without malononitrile


title: Mammary tumor from N7889 (control) mouse [N-9]

city: Toronto

first: Sean

last: Egan

state: Ontario

address: 686 Bay St.

country: Canada

institute: Hospital for Sick Children

description:
R_vs_N_DifExp_FPKMs_DESeq2
R_vs_N_Filt_DifExp_FPKMs_edgeR

library_source: transcriptomic

data_processing:
The quality of the data is assessed using FastQC v.0.11.5
Adaptors are trimmed using Trim Galore v. 0.5.0 and the quality of the trimmed reads is re-assessed with FastQC
The trimmed reads are also screened for presence of rRNA and mtRNA sequences using FastQ-Screen v.0.10.0
The raw trimmed reads are aligned to the reference genome using the STAR aligner, v.2.6.0c.
The filtered STAR alignments are processed to extract raw read counts for genes using htseq-count v.0.6.1p2
Assembly: GRCm38.p6, M25 Gencode gene models
Supplementary files format and content: tab-delimited text file includes raw counts for each sample
Supplementary files format and content: tab-delimited text file includes FPKM values for each sample

molecule: total RNA

source_name: Mammary tumor

extract_protocol:
RNA was isolated from tumors using Qiagen RNeasy Kit (Cat # 74104)
Libraries were prepared using Illumina Stranded Total RNA Prep Ligation with Ribo-Zero Plus  kit following manufacturer's protocol

tag: tissue

value: Mammary tumor

tag: genotype


title: H57, >120 μm, replicate 1, scRNAseq


title: Mammary tumor from N7357 (control) mouse [N-7]


title: mESC(serum)-12_control

description: Control group of CLEVER-seq

value: mESC(serum)


title: Mammary tumor from N7888 (control) mouse [N-8]


title: Mammary tumor from N5829 (control) mouse [N-4]


title: Mammary tumor from N7347 (control) mouse [N-6]


title: H47, >120 μm, replicate 1, scRNAseq


title: mESC(2i)-15_control

description: Control group of CLEVER-seq


title: H43, >120 μm, replicate 1, scRNAseq


title: Mammary tumor from N5759 (control) mouse [N-3]


title: mESC(2i)-16_control

description: Control group of CLEVER-seq


title: Colon, T cell-specific Il22 cKO Infected, rep1

city: Birmingham

first: Min

last: Gao

state: AL

address: 1900 University Blvd, THT Building Suite #130G

country: USA

institute: University of Alabama at Birmingham

data_processing:
Raw data were processed with the Cell Ranger pipeline (v.3.0.2).
Seurat110 (v.3.0.0), implemented using the R package was applied to exclude low-quality cells, identify clusters and differently expression genes.
Assembly: mm10
Supplementary files format and content: feature counts for each Sample
Supplementary files format and content: tab-delimited text files include feature counts for all samples

extract_protocol:
Single cells were captured into Gel Bead-in-EMulsions using a 10x chromium controller.
Single-cell 5’ biased transcriptome libraries were prepared using a 5’ Library & Gel Bead Kit (PN-1000014) according to the 10x Genomics manual

tag: cell line

value: Colon epithelial and intraepithelial cells

value: Il22fl/fl.CD4/Cre cKO Cr D9-1


title: mESC(2i)-14_control

description: Control group of CLEVER-seq


title: H42, >120 μm, replicate 1, scRNAseq


title: Mammary tumor from N4441 (control) mouse [N-1]


title: Colon, Control Infected, rep2

value: Control Cr D9-1


title: Colon, T cell-specific Il22 cKO Infected, rep2


title: Colon, Naïve, rep2


title: H34, >120 μm, replicate 1, scRNAseq


title: TE-02_control

description: Control group of CLEVER-seq

value: TE


title: Colon, Control Infected, rep1


title: mESC(serum)-11_control

description: Control group of CLEVER-seq


title: Colon, Naïve, rep1


title: pig testis

city: Qingdao

first: JunLin

last: Song

address: Changcheng Road

institute: JunLin Song

data_processing:
The demultiplexing, barcoded processing, gene counting and aggregation were made using the Cel Ranger software v2.1.1(https://support 10xgenomics. com/single-cel-gene-expression/software/pipelines/latestwhat- is-cel-range)
Raw count matrices generated by Cell Ranger were imported to Seurat 4.3.0 and filtered for only high-quality cells. Briefly, we removed cells with less than 200 detected genes and genes detected in 3 or fewer cells. Gene expression values were log normalized and scaled before further downstream analyses. Cell clustering and UMAP analysis were performed based on the statistically significant principal components. Marker genes of each cell cluster were determined by Log Fold Change threshold above 0.25 using the default Wilcoxon rank-sum test. After identification of cell clusters, raw count matrices of data subsets were imported to Monocle and only expressed genes above threshold (0.1) were used for analyses. Differentially expressed genes or significantly variable genes among cells were identified by Monocle and used for ordering cells in pseudotime. Only genes with a dispersion ratio above 0.1 were used for training the pseudotime trajectories. To generate the pseudotime heatmaps, DEGs (Differentially Expressed Genes) among cell clusters in pseudotime with qval <0.1 were included and clustered hierarchically based on their expression trends.
Assembly: 10mm
Supplementary files format and content: Tab-separated  values files and matrix files

organism: Sus scrofa

source_name: pig testis

extract_protocol:
The mice and pigs’ testis were cut into pieces. The mice and pigs’ epididymis were collected and divided into epididymis caput, epididymis corpus and epididymis cauda. Cut the fresh tissue into pieces, transfer to the 0.2% collagenase IV and DNAse I digestion solution, and incubate at 37°C for 15 min.
Self-designed using MGI C4 droplet generation device, through efficient mRNA capture magnetic beads and droplet recognition microbeads and single-cell RNA library preparation kit The single-cell (nuclear) suspension can be quickly prepared into the library corresponding to the sequencing platform. The DNBelab C library contains cDNA Library, which are derived from MRNA-capturing magnetic beads and droplet recognition microbeads, respectively.

value: pig testis


title: H33, >120 μm, replicate 1, scRNAseq


title: TE-01_control

description: Control group of CLEVER-seq


title: H55, >120 μm, replicate 1, scRNAseq


title: pig epididymis cauda

source_name: pig epididymis cauda

value: pig epididymis cauda


title: ICM-02_control

description: Control group of CLEVER-seq

value: ICM


title: H31, >120 μm, replicate 1, scRNAseq


title: pig epididymis corpus

source_name: pig epididymis corpus

value: pig epididymis corpus


title: 915_C8

city: Shanghai

first: Guofen

last: Ma

state: Shanghai

address: 280 Chongqing South Road,Huangpu District,

department: School of Medicine

institute: Shanghai Jiao Tong University

data_processing:
Sequenced reads were trimmed for adapter sequences and masked for low-complexity or low-quality sequences, then aligned to GRCm38 (mm10) using a RefSeq annotation GFF file.
Sequence alignment was performed using STAR v2.5.3 in two pass mode.PCR duplicates were masked and removed using the STAR option ‘bamRemoveDuplicates’
Gene counts were computed using the R Genomic Alignments package.
Raw counts data were log-transformed to assess the expression levels of specific genes.
Raw read count table was then used to identify the differentially expressed genes between the Pyr <-- ORBvl and Pyr <-- LP neurons using the DESsq2.
Assembly: mm10
Supplementary files format and content: Text file includes read counts of every gene in each sample
Supplementary files format and content: Text file includes read FPKM values of every gene in each sample

source_name: Pyramidal neuron

extract_protocol:
Following electrophysiological recording, single L5 Pyrs were collected immediately by applying light negative pressure through the same glass patch pipette. Following successful aspiration, the sample was transferred in a volume of 4 μL lysis buffer.Collected single cells were immediately spun down at room temperature and put on dry ice until -80°C storage.
cDNA libraries were produced using a Smart-seq2-based Single Cell Full Length mRNA-Amplification Kit (N712, Vazyme) according to the manufacturer’s instructions. To construct the final sequencing libraries, 1 ng of purified cDNA from each sample was tagmented using an Illumina Nextera XT Library Preparation kit (TD503 Vazyme).

value: Pyramidal neuron

tag: cell subtype

value: Pyr <-- ORBvl

value: WT


title: 919_C3


title: H27, >120 μm, replicate 1, scRNAseq


title: ICM-01_control

description: Control group of CLEVER-seq


title: 916_C5


title: H218, >120 μm, replicate 1, scRNAseq


title: 916_C4


title: G99, 110-119 μm, replicate 1, scRNAseq


title: 915_C3


title: mESC(2i)-13_control

description: Control group of CLEVER-seq


title: 4-cell-embryo3_blastomere-a_control

description: Control group of CLEVER-seq


title: 915_C10


title: G98, 110-119 μm, replicate 1, scRNAseq


title: 914_C5


title: G181-WGS

city: Göttingen

first: Argyris

last: Papantonis

state: Goettingen

address: Robert-Koch-Str. 40

department: Institute of Pathology

country: Germany

institute: University Medical Center Göttingen

description: whole squencing data

data_processing:
WGS read pairs were first mapped to GRCh38 by BWA mem (v0.7.17)
Duplicate reads were removed by Picard (v2.20.7)
WGS-based CNV profiles and segments were calculated via the CNVkit (v0.9.9)77 batch function using the “--segment-method hmm-tumor -m wgs --drop-low-coverage --target-avg-size 25000” parameters.
Assembly: GRCh38
WGS raw data was stored in filtered sorted bam format (GSC181.redup.filted.sorted.bam).
Supplementary files format and content: CNV profiled output generated by CNVkit (v0.9.9) was stored in bedgraph format (GSC181_cnvkit.cnv)
Library strategy: DNA-seq

organism: Homo sapiens

source_name: glioblastoma stem-like cells

extract_protocol:
For WGS DNA was extracted from mouse tails by Salt lysis buffer and Isopropanol DNA precipitation.
For WGS, library was generated using Illumina TrueSeq standard protocol.

value: glioblastoma stem-like cells

tag: assay

value: WGS


title: 2-cell-embryo10_blastomere-b_control

description: Control group of CLEVER-seq


title: G97, 110-119 μm, replicate 1, scRNAseq

value: Oocyte


title: hES cell-derived microglial progenitor (CD14+) in vitro 3 [Progenitor-3]

city: Stanford

first: Yongjin

last: Yoo

address: 265 Campus Drive

department: Institute for Stem Cell Biology and Regenerative Medicine

institute: Stanford University

description:
Progenitor-3
Normalized_counts.txt

data_processing:
150 bp paired-end reads were generated producing a mean of 37.0 million reads per sample.
RNA-sequencing data were processed and interpreted using in-house bioinformatic analysis pipelines in Sherlock computing clusters of The Stanford Research Computing Center.
Briefly, raw sequencing reads were demultiplexed with bcl2fastq and aligned to mouse transcriptome (mm10; GRCm38.p6) using STAR (version 2.5.2b) with the default parameters.
The expression of Gencode-annotated was calculated using HTSeq-count, and FPKM/TPM values were calculated using RSEM with the default parameters.
Raw counts of each gene were normalized, and genes with low counts were removed using the mean of normalized counts as a filter statistic, performed principal component analysis, and conducted differential expression analysis using the DESeq2 package (3.17) in R with standard parameters.
Assembly: hg38
Supplementary files format and content: tab-delimited text file including normalized counts for each sample

source_name: Brain

extract_protocol:
DAPI-negative Human CD45+CD11b+ cells were directly sorted in 400μL TRIzol LS using a FACSAria II. Total RNA was extracted and then purified using a Qiagen RNeasy MinElute Cleanup Kit.
The SMART-Seq v4 Ultra Low Input RNA Kit was used to generate cDNA and libraries according to the manufacturer’s protocol. The libraries were synthesized, indexed libraries were pooled and sequenced on NovaSeq platform.

value: Brain

value: Human microglial progenitor (CD14+) differentiated from H1(WA01)

value: Microglia

value: wildtype


title: 914_C3


title: 914_C2


title: 2-cell-embryo9_blastomere-b_control

description: Control group of CLEVER-seq

value: Control group using H2O without malononitrile


title: pig epididymis caput

source_name: pig epididymis caput

value: pig epididymis caput


title: hES cell-derived microglial progenitor (CD14+) in vitro 2 [Progenitor-2]

description:
Progenitor-2
Normalized_counts.txt


title: G63, 110-119 μm, replicate 1, scRNAseq


title: 4-cell-embryo3_blastomere-b_control

description: Control group of CLEVER-seq


title: 914_C1


title: 2-cell-embryo9_blastomere-a_control

description: Control group of CLEVER-seq


title: G35, 110-119 μm, replicate 1, scRNAseq


title: hES cell-derived microglial progenitor (CD14+) in vitro 1 [Progenitor-1]

description:
Progenitor-1
Normalized_counts.txt


title: 913_C7


title: 2-cell-embryo8_blastomere-b_control

description: Control group of CLEVER-seq


title: G272, 110-119 μm, replicate 1, scRNAseq


title: 915_C5


title: Primary human microglia from epilepsy patient 3 [PrimaryhMG-3]

description:
PrimaryhMG-3
Normalized_counts.txt

value: Primary


title: 913_C6


title: 2-cell-embryo8_blastomere-a_control

description: Control group of CLEVER-seq


title: Primary human microglia from epilepsy patient 2 [PrimaryhMG-2]

description:
PrimaryhMG-2
Normalized_counts.txt


title: G266, 110-119 μm, replicate 1, scRNAseq


title: Primary human microglia from epilepsy patient 1 [PrimaryhMG-1]

description:
PrimaryhMG-1
Normalized_counts.txt


title: 2-cell-embryo10_blastomere-a_control

description: Control group of CLEVER-seq


title: G86, 110-119 μm, replicate 1, scRNAseq


title: G237, 110-119 μm, replicate 1, scRNAseq


title: 909_C3


title: hES cell-derived microglia transplanted into mouse brain after PLX 6 [PLX-2wk-Re-4]

description:
PLX-2wk-Re-4
Normalized_counts.txt

value: hES (WA01; H1) cell-derived microglia transplanted into mouse brain after PLX


title: Paternal PN-13_control

description: Control group of CLEVER-seq

value: Paternal Pronuciel


title: 902_C3


title: G232, 110-119 μm, replicate 1, scRNAseq


title: Paternal PN-12_control

description: Control group of CLEVER-seq


title: G220, 110-119 μm, replicate 1, scRNAseq


title: hES cell-derived microglia transplanted into mouse brain after PLX 5 [PLX-2wk-Re-2]

description:
PLX-2wk-Re-2
Normalized_counts.txt


title: hES cell-derived microglia transplanted into mouse brain after PLX 4 [PLX-2wk-Re-1]

description:
PLX-2wk-Re-1
Normalized_counts.txt


title: Oocyte-14_control

description: Control group of CLEVER-seq


title: G11, 110-119 μm, replicate 1, scRNAseq


title: 902_C2


title: Oocyte-13_control

description: Control group of CLEVER-seq


title: hES cell-derived microglia transplanted into mouse brain after PLX 3 [PLX-0wk-Re-3]

description:
PLX-0wk-Re-3
Normalized_counts.txt


title: 902_C1


title: 831_C4


title: hES cell-derived microglia transplanted into mouse brain after PLX 2 [PLX-0wk-Re-2]

description:
PLX-0wk-Re-2
Normalized_counts.txt


title: 909_C2


title: Paternal PN-14_control

description: Control group of CLEVER-seq


title: Sperm-09_control

description: Control group of CLEVER-seq

value: Sperm


title: F64, 100-109 μm, replicate 1, scRNAseq


title: 831_C3


title: F270, 100-109 μm, replicate 1, scRNAseq


title: 831_C1


title: 831_C2


title: Sperm-08_control

description: Control group of CLEVER-seq


title: F29, 100-109 μm, replicate 1, scRNAseq


title: 902_C5


title: F262, 100-109 μm, replicate 1, scRNAseq


title: OCI-LY3MUT1

city: Vancouver BC

first: Christian

last: Steidl

state: British Columbia

address: 675, West 10th Ave

department: Department of Lymphoid Cancer Research

institute: BC Cancer

data_processing:
Data processing for primary lymphoma and cell line samples was performed as previously described (Ennishi D, Healy S, Bashashati A, et al. TMEM30A loss-of-function mutations drive lymphomagenesis and confer therapeutically exploitable vulnerability in B-cell lymphoma. Nat Med. Apr 2020;26(4):577-588)
For unsupervised analysis, count data was normalized and pre-filtered to keep only genes ≥10 count across all samples (i.e. genes with low expression were removed across all samples).
After the pre-filtering, data was normalized using DESeq2’s (v1.26.0, 32) variance stabilizing transformation and compared between samples for clustering (i.e. TRAF3_status, pathology).
Assembly: GRCh38
Supplementary files format and content: all_libs_vst_counts.txt contains the (VST) normalized read counts for each sample.

extract_protocol:
RNA-seq was performed on three biological replicates for each cell line (DOHH-2, OCI-LY1, HBL-1, OCI-LY3). In brief, total RNA was extracted from 2 x 106 cells (pre-treated with serum-starved culture medium for 12 hr) using a commercial column-based purification kit (Qiagen, Hilden, Germany) with on-column DNase treatment.
Following QC and quantification, 500 ng input RNA was prepped using the TruSeq Stranded mRNA kit and ran on a NextSeq550 instrument (Illumina, San Diego, CA).

value: Diffuse Large B-cell Lymphoma (Activated B-cell-like)


title: OCI-LY3MUT2


title: 830_C7

value: Pyramidal neuron


title: OCI-LY3WT2


title: 830_C3

value: Pyr <-- ORBvl


title: F96, 100-109 μm, replicate 1, scRNAseq


title: F128, 100-109 μm, replicate 1, scRNAseq


title: Oocyte-12_control

description: Control group of CLEVER-seq


title: F92, 100-109 μm, replicate 1, scRNAseq


title: OCI-LY3WT1

value: WT


title: 830_C2


title: F126, 100-109 μm, replicate 1, scRNAseq


title: hES cell-derived microglia transplanted into mouse brain after PLX 1 [PLX-0wk-Re-1]

description:
PLX-0wk-Re-1
Normalized_counts.txt


title: 830_C1


title: HBL1MUT3


title: F123, 100-109 μm, replicate 1, scRNAseq


title: OCI-LY3MUT3


title: 8-27_C4


title: F108, 100-109 μm, replicate 1, scRNAseq


title: 8-27_C3


title: 830_C8


title: F263, 100-109 μm, replicate 1, scRNAseq


title: 8-26_C2


title: E8, 90-99 μm, replicate 1, scRNAseq


title: HBL1MUT1


title: F18, 100-109 μm, replicate 1, scRNAseq


title: E67, 90-99 μm, replicate 1, scRNAseq


title: DOHH2WT2

value: Diffuse Large B-cell Lymphoma (Germinal Centre B-cell-like)


title: DOHH2WT1


title: 8-23_C5


title: E287, 90-99 μm, replicate 1, scRNAseq


title: DOHH2WTBULK


title: E53, 90-99 μm, replicate 1, scRNAseq


title: HBL1WT2


title: E286, 90-99 μm, replicate 1, scRNAseq


title: OCI-LY3WTBULK


title: 8-23_C3


title: E148, 90-99 μm, replicate 1, scRNAseq


title: HBL1WT1


title: 8-23_C2


title: 8-23_C1


title: HBL1WTBULK


title: 822_C1_2


title: HBL1MUT2


title: E152, 90-99 μm, replicate 1, scRNAseq


title: 823_C4_2


title: 8-22_C6


title: OCI-LY1MUT2


title: 8-22_C5


title: E131, 90-99 μm, replicate 1, scRNAseq


title: E132, 90-99 μm, replicate 1, scRNAseq


title: OCI-LY1MUT3


title: OCI-LY1MUT1


title: E127, 90-99 μm, replicate 1, scRNAseq


title: OCI-LY1WT1


title: E112, 90-99 μm, replicate 1, scRNAseq


title: 811_C4


title: E109, 90-99 μm, replicate 1, scRNAseq


title: 811_C3


title: OCI-LY1WT2


title: 8-22_C3


title: OCI-LY1WTBULK


title: DOHH2MUT3


title: D76, 80-89 μm, replicate 1, scRNAseq


title: 811_C1


title: 727_C4


title: 919_C5

value: Pyr <-- LP


title: D78, 80-89 μm, replicate 1, scRNAseq

value: Oocyte


title: DOHH2MUT2


title: DOHH2MUT1


title: D69, 80-89 μm, replicate 1, scRNAseq


title: D73, 80-89 μm, replicate 1, scRNAseq


title: D6, 80-89 μm, replicate 1, scRNAseq


title: HC10_h

city: DAEJEON

first: SEONG EUN

last: LEE

address: 282 MUNHWA-RO

country: South Korea

institute: CHUNGNAM NATIONAL UNIVERSITY

data_processing:
FastQC(v0.11.7) software used for quality check
Trimmomatic(0.38) performs trimming with various parameters for illumina paired-end or single-end.
HISAT2(v2.1.0) quickly and accurately maps high-throughput sequencing data to the reference genome to produce aligned reads.
The transcript assembly through the StringTie(v2.1.3b) program is performed using the information of the aligned reads based on the reference.
The expression profile obtained by transcript quantification of each sample is extracted with FPKM or RPKM, which is the normalization value considering read count and transcript length and depth of coverage.
Assembly: GRCh38
Supplementary files format and content: tsv files of read count of each samples

source_name: normal thyroid

extract_protocol:
RNA was harvested using Trizol reagent. 1ug of total RNA was used for the construction of sequencing libraries.
RNA libraries for RNA-seq were prepared using SMART-Seq v4 Ultra Low Input RNA for Sequencing, TruSeq RNA Library Preparation v2 Kit following manufacturer's protocols.

value: normal thyroid

value: high-density neutrophils


title: D5, 80-89 μm, replicate 1, scRNAseq


title: 916_C3


title: D294, 80-89 μm, replicate 1, scRNAseq


title: 916_C2


title: HC7_h


title: D283, 80-89 μm, replicate 1, scRNAseq


title: HC8_h


title: 919_C2


title: D204, 80-89 μm, replicate 1, scRNAseq


title: HC6_h


title: HC5_h


title: 915_C9


title: HC4_h


title: 915_C7


title: HC2_h


title: 915_C6


title: D2, 80-89 μm, replicate 1, scRNAseq


title: D151, 80-89 μm, replicate 1, scRNAseq


title: 919_C4


title: HC9_h


title: 915_C2


title: D13, 80-89 μm, replicate 1, scRNAseq


title: D2_h

source_name: poorly differentiated thyroid cancer

value: poorly differentiated thyroid cancer


title: 916_C1


title: D121, 80-89 μm, replicate 1, scRNAseq


title: 915_C11


title: D6_h

source_name: follicular thyroid cancer

value: follicular thyroid cancer


title: D120, 80-89 μm, replicate 1, scRNAseq


title: 914_C4


title: 913_C2


title: D1_h

source_name: anaplastic thyroid cancer

value: anaplastic thyroid cancer


title: 915_C1


title: D3_h

source_name: papillary thyroid cancer

value: papillary thyroid cancer


title: D111, 80-89 μm, replicate 1, scRNAseq


title: D115, 80-89 μm, replicate 1, scRNAseq


title: 913_C5


title: C6_h


title: C7_h


title: 913_C4


title: D103, 80-89 μm, replicate 1, scRNAseq


title: HC1_h


title: 913_C3


title: D144, 80-89 μm, replicate 1, scRNAseq


title: C91, 70-79 μm, replicate 1, scRNAseq


title: 913_C1


title: C5_h


title: C9, 70-79 μm, replicate 1, scRNAseq


title: C289, 70-79 μm,  replicate 1, scRNAseq


title: 909_C4


title: 909_C1


title: C2_h


title: C4_h


title: C296, 70-79 μm, replicate 1, scRNAseq


title: C250, 70-79 μm, replicate 1, scRNAseq


title: C244, 70-79 μm, replicate 1, scRNAseq


title: HT-29_treated_LB-100_rep2

city: Warsaw

first: Michal

last: Krzyszton

address: Pawinskiego 5a

country: Poland

institute: Institute of Biochemistry and Biophysics, Polish Academy of Sciences

data_processing:
Sequencing data was demultiplexed into FastQ files using BCLConvert version 3.9.3 (Illumina).
The paired-end reads were trimmed for adapter sequences using SeqPurge version 2019_09 and aligned to the human reference genome using HiSat2 version 2.1.0 with the pre-built grch38_snp_tran reference.
Splicing events were determined using rMats version 4.1.2 using the Ensembl release 100 transcript GTF.
Gene counts were generated using HTseq-count and Homo_sapiens.GRCh38.102.gtf
Assembly: GRCh38 (hg38)
Supplementary files format and content: gene counts in .txt format

growth_protocol: SW-480 and HT-29 were cultured in, respectively, Leibovitz's L-15 Medium and McCoy's 5A Medium supplemented with 10% Fetal Bovine Serum (FBS) and 1% Pennicilin/Streptomycin under humified atmosphere buffered with 5% CO2. Cells were split when subconfluent.

extract_protocol:
For harvesting, the cells were scraped into 15 ml tubes, washed once with cold PBS, and homogenized in RLT buffer (Qiagen #79216). Total RNA was isolated using the RNeasy Mini Kit (74106, Qiagen) including an on-column DNase digestion (79254, Qiagen), according to the manufacturer’s instructions. Quality and quantity of the total RNA was assessed on the 2100 Bioanalyzer instrument following the manufacturer’s instructions “Agilent RNA 6000 Nano” (G2938-90034, Agilent Technologies).
Total RNA samples were subjected to TruSeq stranded mRNA library preparation, according to the manufacturer's instructions (Document # 1000000040498 v00, Illumina). The stranded mRNA libraries were analyzed on a 2100 Bioanalyzer instrument following the manufacturer’s protocol “Agilent DNA 7500 kit” (G2938-90024, Agilent Technologies), diluted to 10 nM and pooled equimolar into multiplex sequencing pools for paired-end sequencing on the NovaSeq 6000 Illumina sequencing instrument.
Paired-end sequencing was performed using 101 cycles for Read 1, 19 cycles for Read i7, 10 cycles for Read i5 and 101 cycles for Read 2, using the NovaSeq6000 SP Reagent Kit v1.5 (200 cycles) (20040719, Illumina).

treatment_protocol: Cells were treated with 4 uM LB-100 PP2A inhibitor for 12 hrs.

value: colorectal adenocarcinoma

value: LB-100 (PP2A inhibitor), for 12h in 4 uM concentration


title: C1_h


title: 907_C4


title: HT-29_treated_LB-100_rep1


title: 909_C5


title: 907_C3


title: Dermal Fibroblasts - RNA-seq - Rep2

city: Rochester

first: Alexandre

last: Gaspar-Maia

state: MN

address: 200 First street SW

institute: Mayo Clinic

data_processing:
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence using TrimGalore v0.6.4 (Cutadapt v3.1)
Reads were then aligned to hg19 (mm9 for DF) whole genome using bowtie2 v2.3.3.1, alignment files were sorted and indexed using samtools v1.9, duplicate removal done using Picard v2.9.0
deepTools bamCoverage v3.5.0 was used to convert bam to bigWig
macs2 v2.2.7.1 was used to call peaks (to convert bam to bed)
scATAC-seq reads were processed using 10x Genomics Cell Ranger ATAC v2.0.0; scMultiome reads were processed using 10x Genomics Cell Ranger ARC v2.0.0; Downstream analysis was done using Seurat and Signac.
Assembly: hg19 (mm9 for DF; hg38 and mm10 for single-cell data)
Supplementary files format and content: bigWig (as produced by deepTools bamCoverage)
Supplementary files format and content: bed (as produced by macs2)

source_name: Dermal Fibroblasts

growth_protocol: Normal Human Melanocytes (NHM) were cultures in Dermal Cell Basal Medium (ATCC) with the addition of 5 µg/mL Insulin, 50 µg/mL Ascorbic Acid, 6 mM L-Glutamine, 1.0 µM Epinephrine, 1.5 mM Calcium Chloride, Peptide Growth Factor and M8 Supplement. MCF-7, DFs and MDA-MB-231L cells were grown in DMEM (Gibco) with 4.5 g/L D-glucose, 110 mg/L sodium pyruvate, 10% FBS and 1% Penicillin/Streptomycin (Hyclone). HMEC cells were grown in complete Mammary Epithelial Cell Growth Media.

extract_protocol:
DNA for ATAC-seq  was prepared from 20,000 cells following the OMNI-ATAC procedures from Corces et al. (Nature Methods 2017) and Buenrostro et al. (Curr Protoc Mol Biol 2016), with modifications, using the Nextera kit (Illumina).  The cells were lysed for 3 minutes on ice and transposed for 30 minutes at 37C.  DNA was cleaned-up using procedures of a DNA extraction column.
The DNA libraries were prepared with 5-9 cycles of PCR amplification with the NEB High Fidelity Master Mix (New England Biolabs).  Clean up was done using the Zymo DNA Clean and Concentrator kit (Zymo Research) and followed with AMPure XP (Beckman Coulter) bead cleanup to remove primer dimers and under-digested chromatin.
Approximately 500,000 DFs were used for total RNA extraction using RNeasy Mini Kit (Qiagen). RNA quality control was performed using Agilent RNA 6000 Nano Kit and all samples had RNA Integrity Number higher than 9.8. Total RNA (1.5 μg) was used for poly(A) mRNA selection using NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB) according to the manufacturer’s protocol
Directional, strand-specific RNA libraries were prepared using NEXTflex Rapid Directional RNA-seq Kit (Bioo Scientific) according to the manufacturer’s protocol. Quality of libraries was analyzed using an Agilent bioanalyzer. Barcoded libraries were multiplexed and subjected to 80 bp single-end sequencing
Approximately 100,000 viable cells  were subjected to Tn5 transposase before proceeding to single cell partitioning into gel beads in emulsion and barcoding following established 10X Genomics protocols. Target cell recovery was ~8000 cells per sample
libraries were constructed following established 10X Genomics protocols and sequenced on HiSeq 4000 (Illumina) before demultiplexing,alignment to the reference genome, and post-alignment quality control
Organoids from mammary stem cells were digested with 0.05% trypsin. Cells were then used for nuclei isolation procedure according to 10X genomics specification
Approximately 5000 nuclei per sample were subjected to transposase assays before proceeding to single cell partitioning into gel beads in emulsion, barcoding, pre-amplification, ATAC library construction, cDNA followed by GEX library construction. ATAC and GEX libraries were sequenced separately on an HiSeq 4000 (Illumina) before demultiplexing, alignment to the reference genome, and post-alignment quality control

treatment_protocol: MCF7 cells were treated with estradiol (E2) treatment MCF-7 cells were grown for 5 days in modified DMEM without phenol-red (Hyclone) with 4.5 g/L D-glucose, 4.0 L-glutamine, 10% charcoal-dextran–stripped FBS, 1% Penicillin/Streptomycin and 1nM of 17-β-estradiol or EtOH.

value: Dermal Fibroblasts


title: Dermal Fibroblasts - RNA-seq - Rep1


title: C24, 70-79 μm, replicate 1, scRNAseq


title: 907_C2


title: C234, 70-79 μm, replicate 1, scRNAseq


title: HT-29_untreated_rep1

value: untreated


title: 907_C1


title: C3_h


title: HT-29_untreated_rep2


title: Liver, INT-787 30 mg/kg, Rep 9

city: Hørsholm

first: Henrik

last: Hansen

address: Hørsholm Kongevej 11

department: Histology & NGS

country: Denmark

institute: Gubra

data_processing:
Reads were aligned to the Genome Reference Consortium Mouse Build 38 (GRCm38) version 89 Ensembl Mus musculus genome using Spliced Transcripts Alignment to a Reference (STAR) version 2.5.2a with default parameters.
Assembly: GRCm38/mm10
Supplementary files format and content: Tab-delimited text file contaning raw counts from each sample

source_name: Liver

extract_protocol:
Liver tissue were collected and snap-frozen in liquid nitrogen. RNA was isolated using the NucleoSpin® kit (MACHEREY-NAGEL).
RNA sequence libraries were prepared with NeoPrep (Illumina) using Illumina TruSeq stranded mRNA Library kit for NeoPrep and sequenced on the NextSeq 500 (Illumina) with NSQ 500 hi-Output KT v2 (75 CYS, Illumina).

value: Liver

value: INT-787 30 mg/kg


title: C23, 70-79 μm, replicate 1, scRNAseq


title: 902_C6

value: Pyramidal neuron


title: Liver, Vehicle, Rep 10

value: Vehicle


title: C233, 70-79 μm, replicate 1, scRNAseq


title: C203, 70-79 μm, replicate 1, scRNAseq


title: SW-480_untreated_rep2


title: SW-480_treated_LB-100_rep1


title: C200, 70-79 μm, replicate 1, scRNAseq


title: 902_C7

value: WT


title: Liver, OCA 30 mg/kg, Rep 9

value: OCA 30 mg/kg


title: 902_C4

value: Pyr <-- LP


title: Liver, INT-787 30 mg/kg, Rep 8


title: Liver, OCA 30 mg/kg, Rep 8


title: C199, 70-79 μm, replicate 1, scRNAseq

value: Oocyte


title: 831_C6


title: Liver, OCA 30 mg/kg, Rep 7


title: Liver, Vehicle, Rep 9


title: 831_C5


title: S7w2_KO_4

city: Helsinki

first: Jouni

middle: Antero

last: Kvist

state: Uusimaa

address: Haartmaninkatu 8

department: Research Program for Molecular Neurology

country: Finland

institute: University of Helsinki

data_processing:
The raw data was filtered with cutadapt (Martin, 2011) to remove adapter sequences, ambiquous (N) and low quality bases (Phred score < 25). We also excluded read pairs that were too short (<25bp) after trimming.
The filtered reads were mapped to the human reference genome (GRCh38) with STAR aligner (Dobin et al., 2013).
Gene expression was counted from read pairs mapping to exons using featureCounts in Rsubreads (Liao, Smyth and Shi, 2019). Duplicates, chimeric and multimapping reads were excluded, as well as reads with low mapping score (MAPQ < 10).
The read count data was analyzed with DESeq2 (Love, Huber and Anders, 2014). We analyzed the effect of RFX6 knockout separately for each developmental stage (stage3, stage5 and stage7).
Assembly: GRCh38
Supplementary files format and content: csv, DESeq2 results and read counts

growth_protocol: The RFX6 mutation was introduced into H1 hESCs using Ribonucleoprotein (RNP) CRISPR-Cpf1 system (Integrated DNA Technologies), with a guide and a mutation template that were designed with Benchling (Biology Software, 2017) (gRNA mutation: TTACACTTTTGGCAAGGAATG).

extract_protocol:
Total RNA was extracted using NucleoSpin Plus RNA kit (Macherey-Nagel).
The library used NEBNext Ultra II Directional RNA kit.
Sequencing was performed using NovaSeq SP 2x100 bp v1.5 chemistry.

treatment_protocol: The hESCs H1 cell-lines were differentiated using the extended 7-stage protocol (stages 1–4 in adherent culture, stage 5 in AggreWell (Stemcell Technologies, #34421), and stages 6 and 7 in suspension culture).

value: ESC derived pancreatic cells

value: mutant

tag: alleles

tag: clone

tag: protocol stage

value: stage7 (week 2)

tag: developmental stage

value: SC-islet


title: C198, 70-79 μm, replicate 1, scRNAseq


title: S7w2_KO_3


title: C147, 70-79 μm, replicate 1, scRNAseq


title: Liver, INT-787 30 mg/kg, Rep 7


title: SW-480_treated_LB-100_rep2


title: C146, 70-79 μm, replicate 1, scRNAseq


title: S7w2_KO_1


title: 830_C4


title: C125, 70-79 μm, replicate 1, scRNAseq


title: Liver, OCA 30 mg/kg, Rep 6


title: Liver, Vehicle, Rep 8


title: 830_C5


title: S7w2_WT_4

value: control


title: C101, 70-79 μm, replicate 1, scRNAseq


title: Liver, INT-787 30 mg/kg, Rep 6


title: SW-480_untreated_rep1

value: colorectal adenocarcinoma


title: B84, 60-69 μm, replicate 1, scRNAseq


title: 8-27_C6


title: S7w2_WT_3


title: S7w2_WT_2


title: B74, 60-69 μm, replicate 1, scRNAseq


title: 8-27_C2


title: Liver, OCA 30 mg/kg, Rep 5


title: B65, 60-69 μm, replicate 1, scRNAseq


title: Liver, Vehicle, Rep 7


title: Liver, INT-787 30 mg/kg, Rep 5


title: 8-27_C1


title: S7w2_WT_1


title: S5_KO_4

value: Endocrine precursors


title: 830_C6


title: S7w2_KO_2


title: 826_C1


title: Liver, Vehicle, Rep 6


title: B299, 60-69 μm, replicate 1, scRNAseq


title: B298, 60-69 μm, replicate 1, scRNAseq


title: 8-23_C6


title: S5_KO_3


title: 8-22_C4


title: B297, 60-69 μm, replicate 1, scRNAseq


title: Liver, INT-787 30 mg/kg, Rep 4


title: S5_KO_2


title: 8-27_C5


title: S5_KO_1


title: 8-22_C2


title: Liver, Vehicle, Rep 4


title: Liver, OCA 30 mg/kg, Rep 4


title: 810_C2


title: Liver, Vehicle, Rep 5


title: B268, 60-69 μm, replicate 1, scRNAseq


title: Liver, OCA 30 mg/kg, Rep 3


title: 810_C1


title: B259, 60-69 μm, replicate 1, scRNAseq


title: S3_KO_4

value: Posterior foregut


title: B258, 60-69 μm, replicate 1, scRNAseq


title: 810_C3


title: S5_WT_4


title: B265, 60-69 μm, replicate 1, scRNAseq


title: S5_WT_3


title: Liver, OCA 30 mg/kg, Rep 2


title: S5_WT_2


title: S3_KO_2


title: B264, 60-69 μm, replicate 1, scRNAseq


title: Liver, INT-787 30 mg/kg, Rep 3


title: B246, 60-69 μm, replicate 1, scRNAseq


title: S5_WT_1


title: 727_C3


title: Liver, Vehicle, Rep 3


title: S3_WT_3


title: B224, 60-69 μm, replicate 1, scRNAseq


title: Liver, Vehicle, Rep 1


title: 727_C2


title: B257, 60-69 μm, replicate 1, scRNAseq


title: Liver, INT-787 30 mg/kg, Rep 2


title: B249, 60-69 μm, replicate 1, scRNAseq


title: 727_C1


title: Liver, Vehicle, Rep 2


title: P310s,Sting-KO, replicate 1

first: Erlin

last: Wang

address: 748 Zhongshan Middle Road, Songjiang District, Shanghai

department: Songjiang Hospital

institute: Shanghai Jiao Tong University School of Medicine

data_processing:
Basecalls were performed using bcl2fastq v2.17 for Novaseq output.
Fastp (version 0.23.1) was used to perform basic statistics on the quality of the raw reads.
HISAT2 (version 2.1.0)
Samtools (version 1.9)
featureCounts (version 2.0.0)
Assembly: GRCm39
Supplementary files format and content: Tab-separated files include raw-count values for each Sample
Supplementary files format and content: Tab-separated files include normalized values for each Sample

source_name: hippocampus

growth_protocol: Animals were maintained in facilities with a 12-h light/dark cycle, an average ambient temperature of 21°C, an average humidity of 48%, and had free access to food and water.

extract_protocol:
Hippocampous were removed, flash frozen on dry ice, and total RNA was extracted using TRIzol reagent (Cat#15596018, ThermoFisher Scientific) according to the manufacturer’s instructions.
Total RNA was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEBNext Ultra RNA Library Prep Kit for Illumina (NEB, USA, Catalog #: E7530L) following manufacturer’s recommendations and index codes were added to attribute sequences to each sample.

value: hippocampus

tag: Sex

value: male

tag: age


title: P310s,Sting-KO, replicate 5

value: female


title: EC Fbxw7 calvarial bone-PLD14, replicate 1, scRNAseq

city: Muenster

first: Hyun-Woo

last: Jeong

state: NRW

department: Dept. Tissue Morphogenesis

institute: Max-Planck-Institute for Molecular Biomedicine

description: BD Rhapsody

data_processing:
The FASTQ format of sequencing raw data were processed with BD Rhapsody WTA Analysis pipeline (version 1.11) on SevenBridges Genomics online platform (SevenBridges).
Assembly: mm10
Supplementary files format and content: st files include UMI counts for each cell barcode and genes

source_name: Calvarial bone

extract_protocol:
Skulls with calvarial lesions (diameter: 1 mm) at PLD14 with their corresponding age-matched controls were harvested, cleaned from attached surrounding tissue and collected in digestion enzyme solution (Collagenase type I and IV, 2 mg/ml). Next, bones were cut into small pieces. Samples were digested for 30 min at 37 °C under gentle agitation. Digested samples were transferred to 70 µm strainers in 50 ml tubes to obtain a single–cell suspension, which was resuspended in blocking solution (1% BSA, 1mM EDTA in PBS without Ca2+/Mg2+), centrifuged at 300×g for 5 mins, washed 2–3 times with ice-cold blocking solution, and filtered through 50 µm strainers. Pellets were resuspended in respective volume of blocking solution. Single cell suspensions were subjected to lineage depletion using lineage cell depletion kit (MACS, cat#130-090-858) following the manufacturer’s instructions. Next, lineage negative cells were depleted by CD45 and CD117 using microbeads (MACS, cat#130-052-301 and cat#130-091-224) from lineage negative (Lin-) bone cells to enrich bone stromal cells.
Single cell suspensions were processed with BD Rhapsody single cell RNA-seq. scRNA-seq libraries were evaluated and quantified by Agilent Bioanalyzer using High sensitivity DNA kit (cat#5067-4626) and Qubit (ThermoFisher Scientific, Cat# Q32851). Individual libraries were diluted to 4 nM and pooled for sequencing. Pooled libraries were sequenced by using High Output kit (150 cycle) (Illumina cat#TG-160-2002) with a NextSeq500 sequencer (Illumina).

value: Calvarial bone

value: bone stromal cells

value: floxed Fbxw7/Cdh5-CreERT2

value: drill hole lesion PLD14


title: S3_KO_1


title: B242, 60-69 μm, replicate 1, scRNAseq

source_name: Oocyte


title: A279, <60 μm, replicate 1, scRNAseq


title: P310s,Sting-KO, replicate 4


title: Liver, INT-787 30 mg/kg, Rep 1

value: Liver

value: INT-787 30 mg/kg


title: S3_WT_4

value: ESC derived pancreatic cells


title: B225, 60-69 μm, replicate 1, scRNAseq

value: Oocyte


title: Liver, OCA 30 mg/kg, Rep 1

value: OCA 30 mg/kg


title: P310s,Sting-KO, replicate 3


title: Calvarial bone-uninjured, replicate 1, scRNAseq

description: BD Rhapsody

value: Bone stromal cells

value: uninjured


title: P310s,Sting-WT, replicate 3


title: P310s,Sting-KO, replicate 2


title: S3_KO_3

value: mutant


title: NC_3

city: NANJING

first: liang

last: wang

address: ZHONGYANGMEN

institute: CHINA PHARMACEUTICAL UNIVERSITY

data_processing:
Illumina Casava1.7 software used for basecalling.
After cluster generation, the library preparations were sequenced on an Illumina Novaseq platform and 150 bp paired-end reads were generated.
Genome_build: ensembl_homo_sapiens_grch38_p12_gca_000001405_27

source_name: HEK293A cells

growth_protocol: Cells were maintained in Dulbecco’s modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (HyClone), 100 U/mL penicillin, and 100 µg/mL streptomycin at 37 °C with 5% CO2 incubation. Cells were seeded into 6-well plates (Corning) 24 h prior to transfection at a density of 300,000 cells per well.

extract_protocol:
Total RNA was prepared from transfection experiments after 72 h using a RNA Isolation Reagent (R701-01, Vazyme). A total amount of 1 µg RNA per sample was used as input material for the RNA sample preparations.
Sequencing libraries were generated using NEBNext UltraTM RNA Library Prep Kit for Illumina (NEB, USA) following manufacturer’s recommendations and index codes were added to attribute sequences to each sample

treatment_protocol: Cells were transfected by using GenJet™ In Vitro DNA Tranfection Reagent (Ver. II) (SignaGen) following the manufacturer’s recommended protocol. For each well of a 6-well plate, 1 μg FEN1 plasmids were transfected and 500 pmoL of DNA probes were transfected 48 h later.

value: HEK293A cells

value: human embryonic kidney epithelial cells

tag: transformation

value: negative control


title: S3_WT_2

value: control

value: Posterior foregut


title: P310s,Sting-WT, replicate 2

value: hippocampus

value: male


title: Single cell RNA-seq - adult tdTomato MPs enriched pituitary gland

city: Vancouver

first: Tully

middle: Michael

last: Underhill

address: 2222 Health Sciences Mall

department: Biomedical Research Centre

institute: University of British Columbia

data_processing:
Samples were demultiplexed using cellranger mkfastq with bclfastq2.
Samples were aligned and output matrices generated using cellranger count for alignment.
cellranger aggregate was used to aggregate multiple samples for combined analysis
Genome_build: mm10 with appended tdTomato and EGFP sequences
Supplementary_files_format_and_content: gene matrices, cell barcode and gene list are included as .mtx, .tsv and .tsv files, respectively

source_name: Quiescent Hic1CreERT2/CreERT2;Rosa26LSLtdTomato/tdTomato MP enriched pituitary gland

growth_protocol: Mice were bred and raised to 8 weeks of age using standard approved animal care committee methodology.

extract_protocol:
FACS purified cells were counted and inspected by hemocytometer before input to the 10X chromium library preparation pipeline and subsequent NGS.
10X Chromium Single Cell 3' v2 or v3

treatment_protocol: Adult animals 8 weeks of age were injected with 2.5mg tamoxifen in 100uL sunflower oil daily for 5 consecutive days and allowed a ten day washout period before experments were performed.

tag: strain

value: pituitary gland

value: Mesenchymal Progenitors


title: B114, 60-69 μm, replicate 1, scRNAseq

city: Celbridge

first: Lais

last: Barbosa Latorraca

address: UCD Lyons Farm

institute: University College Dublin

data_processing:
Trim Galore (version 0.6.6) was used to trim raw sequence reads by removing adapter contamination and poor-quality reads with less than 20 Phred score. Only reads with a minimum 20 bp sequence length after trimming were retained for further processing. The data was mapped to the ARS-UCD 1.2 bovine genome using Hista2. Raw counts were quantified using SeqMonk software (version 11.0.10; Babraham Institute; https://www.bioinformatics.babraham.ac.uk/projects/seqmonk/).
Assembly: ARS-UCD 1.2
Supplementary files format and content: Tab-Delimited Text file containing raw read counts for each sample.

extract_protocol:
PCR tubes containing single oocyte samples snap-frozen in PBS + RLT-Plus Buffer at the collection day were transported in dry ice to the Babraham Institute, UK. Oocyte lysates were thawed and transferred to a 96-well plate and incubated with Dynabeads (MyOne Streptavidin C1, Life Technologies) annealed to Smart-seq2 oligo-dTs to capture polyadenylated mRNA. The remaining lysate containing the DNA was transferred to a new 96 well-plate for a subsequent bisulphite conversion analysis. The beads containing the mRNA were diluted in reverse transcription master mix using SuperScript™ II Reverse Transcriptase (Invitrogen) and reverse transcription was performed to produce cDNA.
cDNA was amplified using KAPA HiFi HotStart ReadyMix (Roche), purified with Ampure XP beads with a 1:0.9 ratio, and eluted into 20 μL of water. Amplified cDNA was quantified using a High Sensitivity DNA Assay chip (Agilent Technologies), and libraries were prepared using the Nextera XT Kit (Illumina) with ~300 pg of cDNA in two different replicates. A total of 179 oocytes were sequenced on the NextSeq500 HighOutput 150 bp Single End (replicate 1) and 75 bp Single End (replicate 2) with a sequence depth of ~2 million reads.

tag: cell type


title: Mouse treated with fed+Veh_Replicate2

status: Public on Apr 10 2024

city: Gwanak-gu

first: Chong-Su

last: Kim

state: --- Select One ---

address: 1 Gwanak-ro, Gwanak-gu

institute: Dongduk Women's University

description: 10 weeks old male mice were IP injected with vehicle

data_processing:
Preprocessing for raw reads was performed to remove low-quality reads and artifacts such as adapter sequences, contaminant DNA, and PCR duplicates.
Assembly: mm10
Supplementary files format and content: xlsx file that contains TPM and FPKM values for each sample

source_name: brain

extract_protocol:
Total RNA was isolated using TRIzol (Invitrogen). RNA samples were treated with DNase (Invitrogen) and then purified using RNeasy mini kit (Qiagen).
RNAseq libraries were generated according to the TruSeq Stranded mRNA sample preparation guide.

value: brain

value: 10 weeks

value: vehicle


title: Mouse treated with fed+Veh_Replicate1


title: Mouse treated with dex+Zj7_Replicate2

description: 10 weeks old male mice were IP injected with Dex+Zj7  (10 mg/kg)

value: Dex+Zj7  (10 mg/kg)


title: Mouse treated with dex_Replicate2

description: 10 weeks old male mice were IP injected with Dex (10 mg/kg)

value: Dex (10 mg/kg)


title: S3_WT_1

value: ESC derived pancreatic cells


title: neurons, sorted, scRNA-seq

city: New York

first: Hao

last: Jin

state: New York

address: 3227 Broadway

institute: Columbia University

description: plate

data_processing:
The barcode demultiplexing, alignment and molecular counting were made using Cell Ranger analysis software
Assembly: mm10
Supplementary files format and content: Tab-separated values files and matrix files

extract_protocol:
For sample 1, we isolated single cells from the cNST as previously described with the following modifications. Briefly, mice were anaesthetized with isoflurane and transcardially perfused with ice-cold carbogenated (95% O2, 5% CO2) NMDG-HEPES-ACSF (93 mM NMDG, 2.5 mM KCl, 1.2 mM NaH2PO4, 30 mM NaHCO3, 20 mM HEPES, 25 mM glucose, 10 mM MgSO4, 1 mM CaCl2, 1 mM kynurenic-acid Na salt, 5 mM Na-ascorbate, 2 mM Thiourea, 3 mM Na-pyruvate, pH 7.4). The brainstems were rapidly extracted and sliced into 300 μm sections containing the cNST using a vibratome (Leica, #VT-1000S) in ice-cold NMDG-HEPES-ACSF solution with continuous carbogenation. The cNSTs were dissected, pooled (from 5 animals), and digested in Trehalose-HEPES-ACSF (92 mM NaCl, 2.5 mM KCl, 1.25 mM NaH2PO4, 30 mM NaHCO3, 20 mM HEPES, 25 mM glucose, 2 mM MgSO4, 2 mM CaCl2, 1 mM kynurenic-acid Na salt, 2.5 wt/vol trehalose, pH 7.4) containing Papain (20 U ml-1, Worthington, #LK003150) and DNase I (25 U ml-1) at 35oC for approximately one hour. Using Pasteur pipettes with progressively narrowing tip diameters, the tissue was triturated in DNase I-containing (25 U ml-1) Trehalose-HEPES-ACSF solution to form single-cell suspension. The dissociated cells were passed through 40 μm filter and resuspended in Resuspension-ACSF (117 mM NaCl, 2.5 mM KCl, 1.25 mM NaH2PO4, 30 mM NaHCO3, 20 mM HEPES, 25 mM glucose, 1 mM MgSO4, 2 mM CaCl2, 1 mM kynurenic-acid Na salt, 0.05% BSA, pH 7.4).
The resulting cell suspension was processed by the Columbia Genome Core to encapsulate and barcode individual cells using the 10X Genomics Chromium system.
For sample 2, we used TRAP2; Ai9 mice that were TRAP-labeled with tdTomato in response to LPS. Cells from the cNST were isolated as described above. The cell suspension was stained with DRAQ5 (Thermoscientific, #62254) and Calcein Violet (Thermoscientific, #C34858) to label viable cells, prior to FACS. A total of 288 tdTomato+ LPS TRAPped cells and 96 tdTomato- cells were sorted into 96-well plates pre-loaded with cell lysis buffer containing 0.1% TritonX-100, SuperaseIN (Ambion, #AM2694), 1 mM dNTP and 1 μM capture primer (i.e. bar-coding).
cDNA was synthesized using Maxima Reverse Transcriptase (Thermoscientific, #EP0753) according to manufacturer’s instruction. cDNA from all the wells/cells was combined, followed by clean-up using Silane beads (Thermoscientific #37002D). Pooled cDNA was amplified using Kapa HotStart Mix with SMART PCR primer (0.2 μM), and then purified using AMPureXP beads (Beckman Coulter Life Sciences #A63880). 0.6 ng of cDNA was used as input to prepare libraries using Nextera XT kit (Illumina #FC-131-1024).


title: A280, <60 μm, replicate 1, scRNAseq

value: Oocyte

tag: experimental group


title: l_GT20-18168_S59

city: BAR HARBOR

first: Dan

last: CORTES

state: Maine

address: 600, Mn Str, 600 main st

institute: The Jackson Lab

data_processing:
Low-quality bases (Q < 30) from the 3’ end of reads were removed and reads with more than 30% low-quality bases (Q < 30) overall were filtered out. Trimmed reads aligned to the reference genome using bowtie2 generating a bam file which wass used by rsem to quantify providing expression counts for genes and isoforms separately
BAM files resulting from the alignments were summarized to genes using the featureCounts algorithm
Expression data was normalized using the VOOM function in the Limma package in R
Differentially expressed genes (>2-fold and <5% false discovery rate) were identified by linear modeling and Bayesian statistics using the Limma package
Genome_build: version 74 of mouse reference genome GRCm38
Supplementary_files_format_and_content: tab-delimited text files include log2 counts per million (CPM) values for each Sample ...

source_name: maturation

growth_protocol: mESC were differentiated into NPC and mature neurons according to the protocol we developed in the article where we used this dataset (Cortes 2022)

extract_protocol:
RNA was isolated using the RNeasy Mini Kit (Qiagen) and processed as per manufacturer’s instructions.
libraries were prepared using the kapa mRNA tranded library kit, according to the manufacturer’s instructions. 2x150, Illumina sequencing

treatment_protocol: Cells were treated with a Dyrk1a inhibitor (ID-8) and/or axons were axotomized

tag: background strain


title: P310s,Sting-WT, replicate 5

value: female


title: h_GT20-18164_S96

value: WSB/EiJ

tag: treatment


title: NT1_hp2_3

value: HEK293A cells

value: human embryonic kidney epithelial cells


title: c_GT20-18159_S98


title: e_GT20-18161_S73

value: ctrl


title: P310s,Sting-WT, replicate 4

tag: tissue


title: A277, <60 μm, replicate 1, scRNAseq

tag: diameter (um)


title: NC_1

tag: cell line

value: negative control


title: Calvarial bone-PLD14, replicate 1, scRNAseq

description: BD Rhapsody

value: Calvarial bone

value: Bone stromal cells

tag: genotype

value: drill hole lesion LPLD14


title: NT1_hp2_2


title: A278, <60 μm, replicate 1, scRNAseq


title: 43_GT20-18132_S93

value: ctrl/cut


title: A276, <60 μm, replicate 1, scRNAseq


title: P310s,Sting-WT, replicate 1

value: hippocampus

value: male


title: Single cell RNA-seq - adult tdTomato MPs enriched choroid plexus

source_name: Quiescent Hic1CreERT2/CreERT2;Rosa26LSLtdTomato/tdTomato MP enriched choroid plexus

value: choroid plexus

value: Mesenchymal Progenitors


title: 32_GT20-16840_S118

value: ctrl/ctrl


title: p_GT20-18172_S107


title: 44_GT20-18134_S77


title: o_GT20-18171_S95


title: Mouse treated with dex_Replicate1

value: brain

value: 10 weeks

value: Dex (10 mg/kg)


title: n_GT20-18170_S63

value: WSB/EiJ


title: m_GT20-18169_S108


title: 35_GT20-16853_S44


title: 1_GT20-16825_S116


title: 34_GT20-16819_S50


title: k_GT20-18167_S102


title: 33_GT20-16846_S24


title: i_GT20-18165_S69


title: 31_GT20-16837_S41


title: 30_GT20-16839_S25


title: g_GT20-18163_S78


title: Mouse treated with dex+Zj7_Replicate1

value: Dex+Zj7  (10 mg/kg)


title: f_GT20-18162_S105


title: d_GT20-18160_S74


title: 28_GT20-16816_S120


title: 26_GT20-16827_S55


title: b_GT20-18158_S67


title: a_GT20-18157_S70


title: 36_GT20-16841_S20


title: j_GT20-18166_S79


title: 42_GT20-18115_S62


title: 24_GT20-16844_S29


title: 22_GT20-16826_S59


title: 19_GT20-16833_S49


title: 7_GT20-16817_S117


title: 18_GT20-16838_S17


title: 39_GT20-16850_S30


title: 15_GT20-16852_S19


title: 13_GT20-16843_S115


title: 14_GT20-16835_S54


title: 32_GT20-16840_S43


title: 13_GT20-16843_S34


title: 41_GT20-18125_S99


title: 12_GT20-16815_S46


title: 29_GT20-16824_S57


title: 28_GT20-16816_S38


title: 37_GT20-16822_S45


title: 23_GT20-16821_S119


title: 23_GT20-16821_S56


title: 9_GT20-16851_S58


title: 20_GT20-16848_S40


title: 21_GT20-16842_S23


title: 4_GT20-16845_S61


title: 8_GT20-16818_S33


title: 27_GT20-16849_S37


title: 16_GT20-16830_S48


title: 10_GT20-16823_S35


title: 1_GT20-16825_S22


title: WSB-ctrl-3_GT20-12228_S15


title: WSB-ctrl-2_GT20-12227_S36


title: 11_GT20-16829_S27


title: 5_GT20-16834_S62


title: 25_GT20-16832_S32


title: 7_GT20-16817_S51


title: 17_GT20-16836_S26


title: 3_GT20-16831_S13


title: WSB-ctrl-1_GT20-12226_S42


title: WSB-ID8-2_GT20-12230_S52


title: B6-ID8-1_GT20-12223_S14


title: WSB-ID8-1_GT20-12229_S47


title: B6-ctrl-2_GT20-12221_S31


title: B6-ID8-3_GT20-12225_S16


title: WSB-ID8-3_GT20-12231_S39


title: B6-ID8-2_GT20-12224_S28


title: 6_GT20-16820_S53


title: Inf_II_sh

city: Donostia

first: Estefania

last: Carrasco-Garcia

state: Guipúzcoa

address: Dr. Begiritsain s/n

department: Oncology

country: Spain

institute: Biodonostia

data_processing:
This project uses the filtering software SOAPnuke developed by BGI independently for filtering.
Data filtering steps: (1) Remove the reads containing the adaptor(adaptor pollution);  (2) Remove reads whose N content is greater than 5%;  (3) Remove low-quality reads (we define reads with bases with a quality score less than 15 as the proportion of total bases in the reads that are greater than 20% as low-quality reads).
The filtered "Clean Reads" are saved in FASTQ format. Version v1.5.2. Parameters -l 15 -q 0.2 -n 0.05
Bowtie2 was used to map the clean reads to the reference gene sequence. Version：v2.2.5. Parameters：-q --sensitive --dpad 0 --gbar 99999999 --mp 1,1 --np 1 --score-min L,0,-0.1 -p 16 -k 200
RSEM was used to calculate the gene expression level of each sample. Version：v1.2.8. Parameters：-p 8 --forward-prob 0 --paired-end
Assembly: GRCh38.p13
Supplementary files format and content: Normalized and raw counts of all 8 samples
Supplementary files format and content: excel document (xlsx)

growth_protocol: The gastric cancer cell line MKN45 was cultured as an adherent monolayer at 37°C and 5% CO2 in RPMI medium (Gibco) supplemented with 10% FBS (Gibco), 100 U/ml penicillin and 100 µg/mL streptomycin. Cultures were regularly tested for Mycoplasma.

extract_protocol:
Total RNA was isolated using trizol (Life Technologies, Carlsbad, CA, USA).
1) mRNA enrichment and purification: Oligo dT Selection (mRNA enrichment): Use oligo dT beads to enrich mRNA with poly A tail. 2) RNA fragment and reverse transcription Fragment the RNA and first-strand cDNA was generated using random N6-primed reverse transcription, followed by a second-strand cDNA synthesis with dUTP instead of dTTP. 3) End repair, add A and adaptor ligation The synthesized cDNA was subjected to end-repair and then was 3' adenylated. Adaptors were ligated to the ends of these 3' adenylated cDNA fragments; 4) PCR amplification. Prior to PCR amplification, the dUTP-marked strand is selectively degraded by Uracil-DNA-Glycosylase(UDG). The remaining strand is amplified to generate a cDNA library suitable for sequencing. Many rounds of PCR amplification were performed to enrich the purified cDNA template using PCR primer; 5) Single strand separation and cyclization Denature the PCR product by heat and the single strand DNA is cyclized by splint oligo and DNA ligase; 6) DNA nanoball synthesis 7) Sequencing on DNBSEQ (DNBSEQ Technology) platform

treatment_protocol: For ANKRD6 silencing by shRNA, cells were infected with lentivirus harboring the TRCN0000158616 shANKRD6 plasmid (Sigma, St. Louis, MO, USA) and the corresponding pLKO.1 puro control plasmid #8453 (pLKO) from Addgene, a gift from Dr. Bob Weinberg. All infections were performed for 6hours.

value: ANKRD6 downregulation by shRNA after lentiviral infection


title: Inf_II_pLKO

value: Control

value: Control plasmid for lentiviral infection


title: Inf_III_sh


title: Inf_0_pLKO


title: Inf_I_pLKO


title: B6-ctrl-1_GT20-12220_S21

value: ctrl


title: Inf_0_sh


title: Inf_I_sh


title: Inf_III_pLKO


title: 2_GT20-16828_S60


title: B6-ctrl-3_GT20-12222_S18


title: Brain Organoids - 10uM DTG + 10uM Folic Acid

city: Houston

first: Robert

middle: M

last: Cabrera

state: TX

address: 1 Baylor Plaza

department: CPEH

institute: Baylor College of Medicine

description:
GeneID GeneSymbol S1_Control S2_10uM_DTG S3_20uM_DTG S4_10uM_DTG+10uM_Folic_Acid S5_20uM_DTG+10uM_Folic_Acid
S4_10uM_DTG+10uM_Folic_Acid

data_processing:
fastq files were aligned to version 79 of mouse reference genome GRCm38 using the Subread aligner in the RSubread package.
BAM files resulting from the alignments were summarized to genes using the featureCounts algorithm
Expression data was normalized using the VOOM function in the Limma package in R
Differentially expressed genes (³1.5-fold and £5% false discovery rate) were identified by linear modeling and Bayesian statistics using the Gene Pattern package
Assembly: GRCm38
Supplementary files format and content: tab-delimited text files include log2 counts per million (CPM) values for each Sample ...

source_name: embryonic

growth_protocol: Stem Cell Technologies Cerebral Organoid kit (Stem Cell Catalog # 08570)

extract_protocol:
RNA was isolated from PFA-fixed organoids using the RNeasy Mini Kit (Qiagen) and processed as per manufacturer’s instructions.
The Takara SMARTer® Stranded Total RNA-Seq Kit v3 - Pico Input Mammalian (p/n 634485) was used to generate Illumina-compatible libraries for sequencing.

treatment_protocol: For DTG exposure experiments, 10 μM DTG, 20 μM DTG, 10 μM DTG, and 10 μM Folic acid or 20 μM DTG and 20 μM Folic acid were added to the maturation media from day 19 until day 20

value: embryonic

value: H9 - WiCell

value: hESC

value: wild-type


title: Brain Organoids - 20uM DTG

description:
GeneID GeneSymbol S1_Control S2_10uM_DTG S3_20uM_DTG S4_10uM_DTG+10uM_Folic_Acid S5_20uM_DTG+10uM_Folic_Acid
S3_20uM_DTG


title: Brain Organoids - Control (non treated)

description:
GeneID GeneSymbol S1_Control S2_10uM_DTG S3_20uM_DTG S4_10uM_DTG+10uM_Folic_Acid S5_20uM_DTG+10uM_Folic_Acid
S1_Control


title: FB-P303

city: Nanjing

first: Xiaokai

last: Fang

state: Jiangsu

address: No.12, Jiangwangmiao Street, Xuanwu District, Nanjing City, Jiangsu Province

institute: Dermatology Hospital of Chinese Academy of Medical Sciences

data_processing:
quality control
reads mapping to the reference genome
quantification of gene expression level
differential expression analysis
enrichment analysis of differentially expressed genes, gene set enrichment analysis
Assembly: hg38
Supplementary files format and content: tab-delimited text files include RPKM values for each Sample

source_name: fibroblast

extract_protocol:
RNA was harvested using Trizol reagent. Illumina Truseq RNA Sample Prep Kit (Cat#FC-122-1001) was used to total RNA extraction.
Total RNA was used as input material for the RNA sample preparations.Briefly, mRNA was purified from total RNA using poly-T oligo-attached magneticbeads. Fragmentation was carried out using divalent cations under elevated temperature inFirst Strand Synthesis Reaction Buffer(5X). First strand cDNA was synthesized usingrandom hexamer primer and M-MuLV Reverse Transcriptase(RNase H-). Second strandcDNA synthesis was subsequently performed using DNA Polymerase I and RNase HRemaining overhangs were converted into blunt ends via exonuclease/polymerase activities.After adenylation of 3’ends of DNA fragments, Adaptor with hairpin loop structure wereligated to prepare for hybridization. In order to select cDNA fragments of preferentially370~420 bp in length, the library fragments were purified with AMPure XP system(Beckman Coulter, Beverly, USA). Then PCR was performed with Phusion High-FidelityDNA polymerase, Universal PCR primers and Index (X) Primer. At last, PCR products werepurified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer2100 system.

value: fibroblast


title: FB-P302


title: FB-D-gal3

value: D-gal


title: Kelly cells, siControl, rep1

city: Chicago

first: Susan

middle: L

last: Cohn

state: IL

address: 5721 S. Maryland Ave.

department: Pediatrics

institute: University of Chicago

data_processing:
Raw read quality for all sequencing data was assessed with fastqc (version 0.11.5)
Trimmomatic (version 0.36) was used to trim leading and trailing bases below quality 3 and remove reads less than 36 base pairs in length
Reads were aligned to GRCh38 using Hisat2 (version 2.1.0).
Reads were counted across exons (-exon flag) using featureCounts of subread (version 1.5.3).
Assembly: hg38
Supplementary files format and content: tab-delimited text files include raw counts for each sample

source_name: Kelly

growth_protocol: Kelly cells were grown at 5% CO2 in RPMI1640 (Life Technologies) supplemented with 10% heat-inactivated FBS, 2 mmol/L l-glutamine, and 1% penicillin/streptomycin.

extract_protocol:
Total RNA was harvested from cells using the TRIzol reagent (Invitrogen). Poly(A) RNA was extracted using the Dynabeads mRNA DIRECT Purification kit following the manufacturer's protocol.
RNA was purified by RNA Clean & Concentrator Kits (Zymo Research) and cDNA libraries were constructed using the SMART® cDNA Library Construction Kit (Takara) following the manufacturer’s instructions.

treatment_protocol: Kelly cells were treated with either control siRNA  or siRNA against METTL3 for 72 hours. siRNA was transiently transfected into Kelly cells using lipofectamine RNAiMax (Invitrogen, 13778075) per manufacturer’s instructions. Cells were incubated with 20 μM siRNA in 6 well plates for 72 hours and collected for analysis. Total RNA was then extracted as below.

value: Kelly

value: neuroblastoma cell line, MYCN-amplified

value: WT

value: siControl


title: Kelly cells, siMETTL3, rep1


title: Kelly cells, siMETTL3, rep3


title: Kelly cells, siMETTL3, rep2


title: Kelly cells, siControl, rep2


title: Kelly cells, siControl, rep3


title: FB-UVB3

value: UVB


title: FB-UVB1


title: FB-E2

tag: source

value: elderly


title: FB-C2

value: children


title: FB-C3


title: FB-ATV3

value: ATV


title: FB-ATV2


title: FB-P301


title: NGP cells, STM, 6D, rep3

source_name: NGP

growth_protocol: SK-N-BE2 and NGP cells were grown at 5% CO2 in RPMI1640 (Life Technologies) supplemented with 10% heat-inactivated FBS, 2 mmol/L l-glutamine, and 1% penicillin/streptomycin.

treatment_protocol: SK-N-BE2 and NGP cells were treated with either STM2457 or DMSO for 6 days. SK-N-BE2 cells were treated with 12uM STM2457 and NGP cells were treated with 1uM STM2457. Total RNA was then extracted as below.

value: NGP


title: FB-ATV1


title: FB-D-gal1


title: Brain Organoids - 20uM DTG + 20uM Folic Acid

description:
GeneID GeneSymbol S1_Control S2_10uM_DTG S3_20uM_DTG S4_10uM_DTG+10uM_Folic_Acid S5_20uM_DTG+10uM_Folic_Acid
S5_20uM_DTG+10uM_Folic_Acid


title: FB-D-gal2


title: NGP cells, STM, 6D, rep2


title: FB-UVB2


title: FB-E3


title: NGP cells, DMSO, 6D, rep1

value: DMSO


title: SK-N-BE2 cells, DMSO, 6D, rep2


title: HiChIP_H3K27Ac_SUPB15

city: Memphis

first: Daniel

last: Savic

state: TN

address: 262 Danny Thomas pl.

department: Pharmaceutical Sciences

institute: St. Jude Children's Research Hospital

data_processing:
H3K27Ac HiChIP was processed using the Dovetail Genomics pipeline junt documented at https://hichip.readthedocs.io/en/latest/index.html. In preparation for peak calling processed HiChIP bam files were transformed into bed files using samtools (v. 1.14) and bedtools (v. 2.30.0) with command “samtools view -h -@ ${NCORE} -F 0x900 sorted.mapped.PT.bam | bedtools bamtobed -i stdin > primary.aln.bed”. Peak calling to define anchor regions of interest was performed using MACS2 (v. 2.1.1) with command “macs2 callpeak -t primary.aln.bed --nomodel --extsize 147 -q 0.05”. Final statistically significant loops were identified utilizing FitHiChIP (v. 11.0) with configuration parameters “IntType=3, BINSIZE=5000, UppDistThr=2000000, UseP2PBackgrnd=0, BiasType=1, MergeInt=1, QVALUE=0.05”.
Assembly: HG19
Supplementary files format and content: bedpe for significant loops called at 5kb resolution used to determine if SNVs were in a looping genomic region
Library strategy: Promoter capture Hi-C

growth_protocol: RPMI 1640 + 10% serum

extract_protocol: Arima H3K27Ac HiChIP (Arima: A101020) was performed according to the manufacturers provided instructions using unspecified proprietary buffers, solutions, enzymes, and reagents. Briefly, cells were fixed as in promoter capture Hi-C above and the amount of fixed cell suspension equal to 15g of cell DNA was used for the HiC reactions as in promoter capture Hi-C above. The samples were then sonicated 25 cycles (30s on/ 30s off) using a Diagenode Bioruptor Plus bath sonicator. Chromatin immunoprecipitation of the sonicated HiC samples was performed by first incubating the samples with 0.2ug of H3K27 antibody (Active Motif 91194) per 1ug of sample overnight. The next day antibody bound chromatin was collected using protein A beads included in the kit. The beads were washed three times with buffer R1, twice with buffer R3, once with buffer LC and then twice with LTE (buffers specified in Arima kit). Samples were eluted before undergoing decrosslinking. Libraries were prepared using Swift Biosciences Accel-NGS 2S Plus DNA Library Kit (Cat # or 21096) following instructions provided by Arima Genomics. The paired-end libraries were sequenced on the Illumina Novaseq to generate >200M 150bp reads. The provided FitHiChIP pipeline was used for analysis to call 5kb resolution loops in relaxed background mode (https://github.com/ay-lab/FitHiChIP)

value: ALL


title: Brain Organoids - 10uM DTG

description:
GeneID GeneSymbol S1_Control S2_10uM_DTG S3_20uM_DTG S4_10uM_DTG+10uM_Folic_Acid S5_20uM_DTG+10uM_Folic_Acid
S2_10uM_DTG

value: embryonic


title: HiChIP_H3K27Ac_SEM

source_name: SEM

value: SEM


title: FB-E1


title: MiaPaCa2_initial_population

city: Ann Arbor

first: Brian

last: Magnuson

state: MI

address: 1500 East Medical Center Drive

institute: University of Michigan

data_processing:
Basecalling was performed using bcl2fastq 2 (Illumina).
Reads were trimmed to the bare sgRNA sequence using cutadapt 4.1. Paired-end mates were trimmed separately using a sequence 5’-adjacent to the sgRNA position within the vector (TATATCTTGTGGAAAGGACGAAACACCG), requiring a minimum match of 18 bases to the sequence amd followed by truncation to 20 bases (relevant cutadapt command parameters: -m 18 -O 18 -l 20 --discard-untrimmed).
Trimmed reads were then combined and aligned using bowtie2 2.4.5 to a reference built from each sgRNA in the library flanked by vector sequences (5’ GTTATCAACTTGAAAAAGTGGCACCG and 3’ CTAGATCTTGAGACAAATGGC). The bowtie2 parameter --norc was used to prevent reverse compliment alignment.
Guide RNA counting was performed for sample alignments using MAGeCK 0.5.9.5
Assembly: hg38
Supplementary files format and content: Sample count files are tab-delimited text files with header and contain sgRNA ID ("sgRNA"), target gene name ("Gene"), and read count (sample name).
Library strategy: CRISPR screen

source_name: MIA PaCa-2

growth_protocol: 75 million MIA PaCa-2 cells were seeded at a density of 5 x 105 cells/mL in 6-well plates containing 2 mL of DMEM, 8mg/mL polybrene and the CRISPR screen library virus.

extract_protocol:
Genomic DNA was isolated using the DNeasy blood and tissue kit (Qiagen) according to the manufacturer’s protocol.
For each condition, sgRNA was amplified from 50mg gDNA using Herculase II Fusion DNA Polymerase (Agilent Technologies), column purified using Select-a-Size DNA Clean & Concentrator kit (Zymo Research), and then gel-purified using 6% Novex TBE gel (Thermo), followed by isolation from the gels with Gel Breaker Tubes and Gel Filters (BioChain). The resulting PCR products then underwent end-repair and A-tail addition followed by New England Biolabs (NEB) adapter ligation. The final library was prepared by enriching adapter-ligated DNA fragments using 2x KAPA HiFi HotStart mix and NEB dual code barcode following the manufacter’s protocol. The libraries were then sequenced on an Illumina NovaSeq 6000 (paired-end, 300 cycles).

treatment_protocol: Spin infection was caried out by centrifugation at 1,200 x g for 45 minutes at 37C. After 24-hour incubation, the media was replaced with fresh DMEM. After a subsequent 24-hour incubation, cells were transferred to T-150 flasks (at a density of 3 wells into 1 T150 flask) containing 20 mL of DMEM containing puromycin at 2mg/mL. After 3 days of selection, cells were seeded into sixteen total T-150 flasks at a density of 5 x 106 cells/flask in 20 mL of DMEM containing either DMSO or 200nM of apilimod. Cells were passaged every 3-4 days and re-seeded back to the original cell density. After 14 days, a pool of 15 million cells from each condition were harvested for genomic DNA.

value: MIA PaCa-2

value: pancreatic ductal adenoarcinoma cells

tag: time

value: 0 days


title: FB-C1

value: fibroblast


title: NGP cells, DMSO, 6D, rep3


title: NGP cells, DMSO, 6D, rep2


title: NGP cells, STM, 6D, rep1


title: SK-N-BE2 cells, STM, 6D, rep3


title: ATAC_NALM6_rs1247117_AtoG_clone2_2

data_processing:
Data were analyzed as in (https://doi.org/10.1038/s41375-022-01685-z), GSE161501, and GSE129066.
Assembly: HG19
Supplementary files format and content: .bw for coverage
Supplementary files format and content: .narrowpeak for accessible regions

extract_protocol: Fast-ATAC in fresh primary ALL cells from patients and in a subset of ALL cell lines (BALL1, CEM, Jurkat and P12-Ichikawa) was performed on 10,000 cells as described in (https://doi.org/10.1038/ng.3646) and (https://doi.org/10.1038/s41375-022-01685-z). Paired-end Illumina next-generation sequencing of Fast-ATAC libraries was performed at the Hartwell Center for Bioinformatics and Biotechnology at St. Jude Children’s Research Hospital. Data were analyzed as in (https://doi.org/10.1038/s41375-022-01685-z).


title: ATAC_NALM6_parental_3


title: SK-N-BE2 cells, DMSO, 6D, rep1


title: SK-N-BE2 cells, STM, 6D, rep1


title: MiaPaCa2_DMSO_14d

value: 14 days


title: SK-N-BE2 cells, DMSO, 6D, rep3

value: neuroblastoma cell line, MYCN-amplified


title: MiaPaCa2_apilimod_14d

value: apilimod 200nM


title: ATAC_NALM6_parental_2


title: HiChIP_H3K27Ac_BALL1


title: HiChIP_H3K27Ac_CEM

source_name: CEM

value: CEM


title: HiChIP_H3K27Ac_REH

source_name: REH

value: REH


title: shHMGB1_2

city: Wuhan

first: Dong

last: Chen

state: Hubei

address: 388 GaoXin 2nd Road, East Lake Hi-Tech Development Zone

department: Center for Genome Analysis

institute: ABLife, Inc.

data_processing:
Raw reads containing more than 2-N bases were first discarded. Then adaptors and low-quality bases were trimmed from raw sequencing reads using FASTX-Toolkit (Version 0.0.13).
The short reads less than 16 nt were also dropped. After that, clean reads were aligned to the GRCh38 genome by Tophat2 allowing 4 mismatches. Uniquely mapped reads were used to calculate reads number and FPKM value (fragments per kilobase of transcript per million fragments mapped) for each gene.
The DESeq2 was utilized to screen out the differentially expressed genes (DEGs). A false discovery rate <0.05 and fold change>1.5 or < 2/3 were set as the cut-off criteria for identifying DEGs.
To predict the gene function and calculate the functional category distribution frequency, Gene Ontology (GO) analyses and enriched KEGG pathway were identified using KOBAS 2.0 server. Hypergeometric test and Benjamin-Hochberg FDR controlling procedure were used to define the enrichment of each pathway (corrected p-value<0.05).
Assembly: GRCh38
Supplementary files format and content: tab-delimited text files include reads counts for each Sample
Supplementary files format and content: tab-delimited text files include FPKM values for each Sample

source_name: HeLa cells

extract_protocol:
Total RNA was extracted by the TRIZOL (Ambion). The RNA was further purified with two phenol-chloroform treatments and then treated with RQ1 DNase (Promega) to remove DNA. The quality and quantity of the purified RNA were redetermined by measuring the absorbance at 260 nm/280 nm (A260/A280) using Smartspec Plus (Bio-Rad). The integrity of RNA was further verified by 1.5% agarose gel electrophoresis.
For each sample, 1μg of total RNA was used for RNA-seq library preparation by VAHTS Stranded mRNA-seq Library Prep Kit (Vazyme). Polyadenylated mRNAs were purified and fragmented, and then converted into double strand cDNA. After the step of end repair and a tailing, the DNAs were ligated to VAHTS RNA Adapters (Vazyme). Purified ligation products corresponding to 200-500 bp were digested with heat-labile UDG, and the single strand cDNA was amplified, purified, quantified, and stored at -80℃ before sequencing.For high-throughput sequencing, the libraries were prepared following the manufacturer's instructions and applied to Illumina HiSeq X Ten system for 150 NT paired-end sequencing.

value: HeLa cells

value: transfected with HMGB1 shRNA


title: HiChIP_H3K27Ac_RS411


title: NC_empty vector 1

value: transfected with empty vector


title: NC_empty vector 2


title: 17897X9 EPHB2-KO-4

city: Salt Lake City

first: Patrice

last: Mimche

state: Utah

address: 26 North Medical Dr

institute: University of Utah

data_processing:
The human GRCh38 genome and gene annotation files were downloaded from Ensembl release 98
Trimmed reads were aligned to the reference database using STAR version 2.7.2c in two pass mode to output a BAM file sorted by coordinates
Mapped reads were assigned to annotated genes using featureCounts version 1.6.3
Differentially expressed genes were identified using a 5% false discovery rate with DESeq2 version 1.26.0
Assembly: hg38; H_sapiens_Dec_2013; GRCh38
Supplementary files format and content: FeatureCount output files combined into a single tab-delimited matrix

source_name: skin

growth_protocol: Dermal fibroblasts lines were cultured in the fibroblast growth kit-low serum (ATCC, VA) constituted with the following ingredients: Fibroblast basal medium, ascorbic acid, fetal bovine serum, rhFGF-basic, L-glutamine, and hydrocortisone.

extract_protocol:
Total RNA was extracted using RNA Stat60® (Tel-Test Inc, USA) following standard phenol-chloroform procedure, and further purified using the NucleoSpin RNA-II purification kit (Machery-Nagel) following the manufacturer’s instructions.
Total RNA samples (100-500 ng) were hybridized with Ribo-Zero Gold to substantially deplete cytoplasmic and mitochondrial rRNA from the samples.  Stranded RNA sequencing libraries were prepared as described using the Illumina TruSeq Stranded Total RNA Library Prep Gold kit (20020598) with TruSeq RNA UD Indexes (20022371).  Purified libraries were qualified on an Agilent Technologies 4150 TapeStation using a D1000 ScreenTape assay (cat# 5067-5582 and 5067-5583).  The molarity of adapter-modified molecules was defined by quantitative PCR using the Kapa Biosystems Kapa Library Quant Kit (cat#KK4824).  Individual libraries were normalized to 1.30 nM in preparation for Illumina sequence analysis.  Sequencing libraries (1.3 nM) were chemically denatured and applied to an Illumina NovaSeq flow cell using the NovaSeq XP chemistry workflow (20021665).  Following transfer of the flowcell to an Illumina NovaSeq instrument, a 2 x 151 cycle paired end sequence run was performed using a NovaSeq S2 reagent Kit (20012860).

treatment_protocol: Human dernal fibroblasats (70% confluency) were transfected with EPHB2 Mission®-esiRNA (Sigma Aldrich, USA) delivered into the cells using the lipofectamine® RNAiMAX (Invitrogen, USA) according to the manufacturer’s instruction. After 48H incubation, transfected fibroblast were stimulated with recombinant human TGF-β1 at 10 ng/ml for another 24H.

value: skin

value: Primary cell

value: Human dermal fibroblast

value: Knock out


title: 17897X10 EPHB2-KO-5


title: HiChIP_H3K27Ac_Nalm6

value: ALL


title: HiChIP_H3K27Ac_697


title: PDXMPRALibCD10cyclesPCR2

description: Plasmid DNA

library_source: other

data_processing:
Following next-generation sequencing, the MPRA sequence data was trimmed to contain only barcode sequences without allowing for any mismatches and read counts were determined for all barcodes. To identify significant allele-specific effects mpralm (5) was performed on RNA and DNA barcode counts after merging RNA or DNA counts across all barcodes for each allele.
Assembly: .txt file containing SNV-barcode associations
Supplementary files format and content: .txt file containing SNV-barcode associations
Supplementary files format and content: .txt file containing analysis of all cell lines x all SNVs MPRA data
Library strategy: MPRA

molecule: other

organism: synthetic construct

source_name: PDX MPRA library control

extract_protocol:
MPRA Oligo design
Oligo libraries were designed by following previous work with modified protocols (1-4). MPRA oligos ordered from Agilent (230 bp) were structured as follows: 5’-Primer1-enh-Kpnl-Xbal-barcode-primer2-3’ where primer1 and primer2 are universal primer sites, enh denotes the 175bp variant containing region to test for enhancer activity, Kpnl and Xbal denote recognition sequences for cut sites, and barcode denotes 10-bp tag sequence (see Sup File 6). Agilent oligos were resuspended in 100 ul nuclease free water. All 10-bp barcodes for each variant allele used in MPRA are provided in Supplemental File 7.
MPRA Plasmid Cloning-Input (DNA) Library construction
For plasmid cloning, oligo libraries were amplified by 20 cycles of emulsion PCR (Micellula DNA Emulsion & Purification Kit #E3600, EURx Molecular Biology Products) using Herculase II fusion DNA polymerase (#600675, Agilent), forward and reverse primers (see Sup File 3) to introduce SfiI restriction enzyme sites (GGCCNNNNNGGCC) (NEB) and homology arms to the pMPRA1 plasmid (Cat: #49349, Addgene). Purified PCR products were separated on a 2-4% agarose gel to verify the expected amplification size of 281bp. The pMPRA1 backbone vector was Sfil digested overnight and size selected on a 1% agarose gel. Vector backbone gel extraction was done with the Qiagen gel extraction kit and QIAquick PCR purification kit (28706X4 and 28104).  Gibson assembly was used to clone oligos into vector using 79 ng of inserts, 100 ng of digested vector, and 20 ul Gibson assembly 2x master mix (# E2611S, NEB). The reaction was purified using MinElute PCR purification column (Qiagen), and drop analysis was performed with Millipore filters (Type VSWP 0.25 um Millipore #VSWP02500). For the transformation step, we aimed to obtain 10x CFU bacterial cells than the distinct promoter-tag combinations (unique sequences) in the oligo library. We transformed the Gibson assembly reactions into MegaX DH10B electrocompetent bacteria (#C6400-03, Invitrogen) using GenePulser II electroporator (Bio-Rad). Plasmids were extracted with Qiagen Maxi Prep Kit. For quality control studies, an aliquot of the isolated plasmid library was digested with SfiI and run on 1 % agarose gel to confirm the presence of inserts. To generate linear enhancer-barcode backbone sequences for reporter insertion, 2 ug of plasmid was digested with KpnI/XbaI. A minimal promoter + truncated eGFP was then ligated to the linearized enhancer-barcode backbone and purified using the Qiagen MilEute PCR purification kit. The ligation was then transformed into 1 vial of MegaX electrocompetent bacteria (#C6400-03, Invitrogen) as before. Plasmids were then extracted using a Qiagen Maxiprep kit as before and the elution was verified as a single size band by gel electrophoresis.
MPRA library transfection and sequencing
MPRA plasmid library (10 µg) transfections were done in 10 ALL cell lines having at >95% cell viability (45 million cells x 4 replicates x 10 cell lines) using electroporation with the Neon Transfection system (Thermofisher). Next day, RNA-was harvested using the RNeasy plus mini kit (Cat: #74134, Qiagen) using 4 columns per sample. Once the RNA was isolated, we performed additional DNase digestion using RQ1 RNase-free DNase (Cat: # M6101, Promega). All tubes from the same replicates were combined and added 1 volume of 70% ethanol to the combined lysate and mixed well by pipetting. The DNase treated RNA was again purified with RNeasy mini kit and eluted in 60 ul RNase free water. Total RNA was quantified using DeNovix Ds-11 FX instrument. We yielded 16µg-112µg of total RNA from each replicate depending on the cell line used. mRNA purification was performed using Dynabeads mRNA purification kit (Cat: #61006, Invitrogen). mRNA concentration was measured using Qubit HS RNA (Cat: #Q32852, Invitrogen). We yielded from 0.75-2 ug of mRNA in average from each replicate. cDNA was synthesized using three primers (2 uM) cDNA P1, cDNA P4, and cDNA 6, with the Superscript III first-strand synthesis system (Cat: #18080051, Invitrogen) (see Sup File 6).
Final multiplexing of 50ng cDNA and input plasmid DNA (4 aliquots of MPRA plasmid pool that were independently prepared for next-generation sequencing) was carried out using Q5 Hot Start 2x Master Mix (NEB #M0494S), index primers, and Multiplexing primer 1 for 15 cycles of PCR. The reactions were size selected using AMPure XP beads (Cat: #A63881, Beckman Coulter, Indianapolis, IN) and eluted in 20 ul nuclease free water. The final library concentration was measured using Qubit DNA HS. 20-40 ng of each library was sequenced on the Illumina NovaSeq (200 million x 150bp paired-end reads per sample) at the Hartwell Center for Bioinformatics and Biotechnology at St. Jude Children’s Research Hospital.

value: PDX MPRA library control


title: PDXMPRALibCD6cyclesPCR2

description: Plasmid DNA


title: ATAC_NALM6_rs1247117_AtoG_clone2_3


title: 17897X5 EPHB2-WT-5

value: Wild type


title: ATAC_NALM6_rs1247117_AtoG_clone2_1


title: PDXMPRALibCD10cyclesPCR1

description: Plasmid DNA


title: ATAC_NALM6_rs1247117_AtoG_clone1_3


title: SK-N-BE2 cells, STM, 6D, rep2

value: WT


title: ATAC_NALM6_rs1247117_AtoG_clone1_1


title: ATAC_NALM6_rs1247117_AtoG_clone1_2


title: 17897X2 EPHB2-WT-2


title: ATAC_NALM6_parental_1


title: PDXMPRA1137Rep4

source_name: PDX

value: PDX


title: NC_empty vector 3


title: shHMGB1_1


title: shHMGB1_3


title: 17897X8 EPHB2-KO-3


title: 17897X7 EPHB2-KO-2


title: 17897X4 EPHB2-WT-4


title: 17897X6 EPHB2-KO-1


title: rep3_prep2_MPRALibC6cyclesPCR

description:
Plasmid DNA
barcodes.txt

data_processing:
Following next-generation sequencing, the MPRA sequence data was trimmed to contain only barcode sequences without allowing for any mismatches and read counts were determined for all barcodes. To identify significant allele-specific effects mpralm (69) was performed on RNA and DNA barcode counts after merging RNA or DNA counts across all barcodes for each allele.
Assembly: .txt file containing SNV-barcode associations
Supplementary files format and content: .txt file containing SNV-barcode associations
Supplementary files format and content: .txt file containing analysis of all cell lines x all SNVs MPRA data
Library strategy: MPRA

source_name: MPRA library control

extract_protocol: MPRA Oligo design :Oligo libraries were designed by following previous work with modified protocols (1-4). MPRA oligos ordered from Agilent (230 bp) were structured as follows: 5'-Primer1-enh-Kpnl-Xbal-barcode-primer2-3' where primer1 and primer2 are universal primer sites, enh denotes the 175bp variant containing region to test for enhancer activity, Kpnl and Xbal denote recognition sequences for cut sites, and barcode denotes 10-bp tag sequence (see Sup File 6). Agilent oligos were resuspended in 100 ul nuclease free water. All 10-bp barcodes for each variant allele used in MPRA are provided in Supplemental File 7. MPRA Plasmid Cloning-Input (DNA) Library construction: For plasmid cloning, oligo libraries were amplified by 20 cycles of emulsion PCR (Micellula DNA Emulsion & Purification Kit #E3600, EURx Molecular Biology Products) using Herculase II fusion DNA polymerase (#600675, Agilent), forward and reverse primers (see Sup File 3) to introduce SfiI restriction enzyme sites (GGCCNNNNNGGCC) (NEB) and homology arms to the pMPRA1 plasmid (Cat: #49349, Addgene). Purified PCR products were separated on a 2-4% agarose gel to verify the expected amplification size of 281bp. The pMPRA1 backbone vector was Sfil digested overnight and size selected on a 1% agarose gel. Vector backbone gel extraction was done with the Qiagen gel extraction kit and QIAquick PCR purification kit (28706X4 and 28104).  Gibson assembly was used to clone oligos into vector using 79 ng of inserts, 100 ng of digested vector, and 20 ul Gibson assembly 2x master mix (# E2611S, NEB). The reaction was purified using MinElute PCR purification column (Qiagen), and drop analysis was performed with Millipore filters (Type VSWP 0.25 um Millipore #VSWP02500). For the transformation step, we aimed to obtain 10x CFU bacterial cells than the distinct promoter-tag combinations (unique sequences) in the oligo library. We transformed the Gibson assembly reactions into MegaX DH10B electrocompetent bacteria (#C6400-03, Invitrogen) using GenePulser II electroporator (Bio-Rad). Plasmids were extracted with Qiagen Maxi Prep Kit. For quality control studies, an aliquot of the isolated plasmid library was digested with SfiI and run on 1 % agarose gel to confirm the presence of inserts. To generate linear enhancer-barcode backbone sequences for reporter insertion, 2 ug of plasmid was digested with KpnI/XbaI. A minimal promoter + truncated eGFP was then ligated to the linearized enhancer-barcode backbone and purified using the Qiagen MilEute PCR purification kit. The ligation was then transformed into 1 vial of MegaX electrocompetent bacteria (#C6400-03, Invitrogen) as before. Plasmids were then extracted using a Qiagen Maxiprep kit as before and the elution was verified as a single size band by gel electrophoresis. MPRA library transfection and sequencing MPRA plasmid library (10 µg) transfections were done in 10 ALL cell lines having at >95% cell viability (45 million cells x 4 replicates x 10 cell lines) using electroporation with the Neon Transfection system (Thermofisher). Next day, RNA-was harvested using the RNeasy plus mini kit (Cat: #74134, Qiagen) using 4 columns per sample. Once the RNA was isolated, we performed additional DNase digestion using RQ1 RNase-free DNase (Cat: # M6101, Promega). All tubes from the same replicates were combined and added 1 volume of 70% ethanol to the combined lysate and mixed well by pipetting. The DNase treated RNA was again purified with RNeasy mini kit and eluted in 60 ul RNase free water. Total RNA was quantified using DeNovix Ds-11 FX instrument. We yielded 16µg-112µg of total RNA from each replicate depending on the cell line used. mRNA purification was performed using Dynabeads mRNA purification kit (Cat: #61006, Invitrogen). mRNA concentration was measured using Qubit HS RNA (Cat: #Q32852, Invitrogen). We yielded from 0.75-2 ug of mRNA in average from each replicate. cDNA was synthesized using three primers (2 uM) cDNA P1, cDNA P4, and cDNA 6, with the Superscript III first-strand synthesis system (Cat: #18080051, Invitrogen) (see Sup File 6). Final multiplexing of 50ng cDNA and input plasmid DNA (4 aliquots of MPRA plasmid pool that were independently prepared for next-generation sequencing) was carried out using Q5 Hot Start 2x Master Mix (NEB #M0494S), index primers, and Multiplexing primer 1 for 15 cycles of PCR. The reactions were size selected using AMPure XP beads (Cat: #A63881, Beckman Coulter, Indianapolis, IN) and eluted in 20 ul nuclease free water. The final library concentration was measured using Qubit DNA HS. 20-40 ng of each library was sequenced on the Illumina NovaSeq (200 million x 150bp paired-end reads per sample) at the Hartwell Center for Bioinformatics and Biotechnology at St. Jude Children's Research Hospital. MPRA sequencing analysis Following next-generation sequencing, the MPRA sequence data was trimmed to contain only barcode sequences without allowing for any mismatches and read counts were determined for all barcodes. To identify significant allele-specific effects mpralm (5) was performed on RNA and DNA barcode counts after merging RNA or DNA counts across all barcodes for each allele.  1. P. Kheradpour et al., Systematic dissection of regulatory motifs in 2000 predicted human enhancers using a massively parallel reporter assay. Genome Res 23, 800-811 (2013).  2. A. Melnikov et al., Systematic dissection and optimization of inducible enhancers in human cells using a massively parallel reporter assay. Nature biotechnology 30, 271-277 (2012).  3. A. Melnikov, X. Zhang, P. Rogov, L. Wang, T. S. Mikkelsen, Massively parallel reporter assays in cultured mammalian cells. JoVE (Journal of Visualized Experiments), e51719 (2014).  4. J. Choi et al., Massively parallel reporter assays of melanoma risk variants identify MX2 as a gene promoting melanoma. Nat Commun 11, 2718 (2020).  5. L. Myint, D. G. Avramopoulos, L. A. Goff, K. D. Hansen, Linear models enable powerful differential activity analysis in massively parallel reporter assays. BMC Genomics 20, 209 (2019).

value: MPRA library control


title: Patient 26 TCR Pre-Denosumab TP1

city: San Francisco

first: Mark

last: Bridge

state: CA

address: 513 Parnassus Ave

institute: UCSF

description: bulk TCR-seq

data_processing:
mixcr-3.0.13 was used to analyze amplicons for each sample R1 R2 with flags: --species hs --starting-material rna --5-end v-primers --3-end j-primers --adapters adapters-present
TRB output files were collected and matched against UMIs extracted from the input fastq sequences, and combined with the TRB data into a TRB with UMI file (TRB_w_umi.txt file) for each TRB
Assembly: N/A (raw reads used)
Supplementary files format and content: TRB.txt - TCR amplicon clonotype mixcr analysis file
Supplementary files format and content: TRB_w_umi.txt - TRB with UMI
Library strategy: VDJ-seq

source_name: blood

extract_protocol:
Cryopreserved PBMCs were thawed and 250,000 cells were extracted for RNA using the Invitrogen RNAqueous-Micro Total RNA Isolation Kit (Thermo Fisher #AM1931).
10.4ng of RNA was used for reverse transcription (RT) and cDNA generation, following the Smart-seq2 protocol with modifications. The oligo-dT primer was biotinylated at the 5’ end. A barcode, unique molecular modifiers, and biotin were added to the 5’ end of the TSO primer. Maxima H Minus Reverse Transcriptase (Thermo Fisher #EP0753) was used for reverse transcription. Barcoded RT product was then pooled together using a DNA Clean & Concentrator-5 Kit (Zymo Research, #D4013). 16 cycles were used for cDNA amplification. cDNA was then enriched for alpha and beta TCR receptors separately and used for Illumina library generation.
Modified Smartseq-2 with UMIs and barcodes and targeted TCR alpha and beta amplification.

tag: individual

value: Patient 26

value: blood

value: Immune cells

tag: library type

value: VDJ

value: No Denosumab TP1

tag: additional treatment

value: No Chemo No Steroid


title: Patient 25 TCR Post-Denosumab TP4

description: bulk TCR-seq

value: Patient 25

value: Denosumab TP4


title: PDXMPRA4420Rep2


title: PDXMPRA4420Rep3


title: PDXMPRA4420Rep4


title: PDXMPRALibCD6cyclesPCR1

description: Plasmid DNA