title: PFUnDA-3

first: Hui

last: Yang

address: 7 Panjiayuan Nanli, Room314

department: Department of Toxicology

institute: China National Center for Food Safety Risk Assessment (CFSA)

description:
Library name: sample9
PFUnDA-3

data_processing:
Raw data (raw reads) of fastq format were firstly processed through fastp software.
Reference genome and gene model annotation files were downloaded from genome website directly. Index of the reference genome was built using Hisat2 v2.0.5 and paired-end clean reads were aligned to the reference genome using Hisat2 v2.0.5.
The mapped reads of each sample were assembled by StringTie (v1.3.3b) (Mihaela Pertea.et al. 2015) in a reference-based approach.
featureCounts v1.5.0-p3 was used to count the reads numbers mapped to each gene. And then FPKM of each gene was calculated based on the length of the gene and reads count mapped to this gene.
Differential expression analysis of two conditions/groups (three biological replicates per condition) was performed using the DESeq2 R package (1.20.0). DESeq2 provide statistical routines for determining differential expression in digital gene expression data using a model based on the negative binomial distribution. The resulting P-values were adjusted using the Benjamini and Hochberg’s approach for controlling the false discovery rate . Genes with an adjusted P-value <=0.05 found by DESeq2 were assigned as differentially expressed.
Assembly: hg38
Supplementary files format and content: tab-delimited text files include FPKM values for each sample

source_name: liver

growth_protocol: Human hepatocyte organoid lines were obtained from Beijing Daxiang Biotech. The hepatic organoids were mixed with proliferation medium (Daxiang Biotech, HG100101) containing collagen at a 1:2 ratio (v/v), and 50 μL of the mixture (approximately 500–1,000 cells per well) was seeded into 48-well plates. After solidification at 37°C for 10 minutes, 300 μL of proliferation medium supplemented with 1% anti-apoptotic factor (Daxiang Biotech, IA100101) was added to each well.

extract_protocol:
Total RNA was extracted from  cultured cells withTRIzol reagent.mRNA was purified from total RNA using poly-T oligo-attached magnetic beads.
Fragmentation was carried out using divalent cations under elevated temperature in First Strand Synthesis Reaction Buffer(5X). First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase(RNase H-). Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3’ ends of DNA fragments, Adaptor with hairpin loop structure were ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 370~420 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. At last, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system.

treatment_protocol: Hepatocyte organoids were cultured in a 24 - well plate until they reached a diameter of 100 - 200 micrometers. Then, the culture medium was replaced with differentiation medium. After 5 days of differentiation, they were exposed to PFOA (100μM) and PFUnDA (100μM) for 48 hours.

tag: tissue

value: liver

value: hepatic organoids

value: Liver

value: 100  μM PFUnDA


title: LN, CD8, KP, WT, RNA

status: Public on Jul 07 2026

type: SRA

city: Paris

first: Nathan

last: Vaudiau

address: 25 rue du Docteur Roux

department: Immunology

country: France

institute: Institut Pasteur

description:
Library name: LN_CD8_KP_WT_RNA
10x genomics
RNA

library_source: transcriptomic single cell

data_processing:
Raw sequencing reads were processed using the 10x Genomics software Cellranger. To obtain a cell count matrix, reads were mapped to the mouse mm10 reference genome. scRNA-seq analysis was performed using Seurat v4. For each dataset, cells with low genes per cell or with a high percentage of mitochondrial genes were removed from downstream analyses. Following data QC we obtained 4421 cells from the lungs KP CD8+ T cells dataset, 1404 cells for the tdLN KP WT CD8+ T cells dataset, 3340 cells for the tdLN KP CD11cIRF4 CD8+ T cells dataset, 2076 cells for the tdLN KP WT CD11c+MHCII+ cells dataset and 3150 cells for the tdLN KP CD11cIRF4 CD11c+MHCII+ cells dataset. RNA and ADT expression were normalized separately using the default Seurat LogNormalize and Centered Log Ratio (CLR) approaches, respectively. Top 2000 most variable genes were then determined by applying a VST (Variance Stabilizing Transformation). Datasets were analyzed separately or were integrated together before analysis. For integration, top 2000 most variable genes across datasets were used as anchors with the LogNormalize method. Dimensionality reduction was performed using PCA and the top informative principle components were determined after visualization with an elbow plot and dimensional reduction heatmaps, and used for the non-linear dimensionality reduction technique UMAP. Louvain clustering of the data was performed across a range of resolution parameters visualized using a clustree. Cluster of cells with low number of genes and in which no T lymphocyte cell identifying genes could be detected were not considered in the analysis. Subsequent analyses were performed using Seurat default parameters.
Assembly: mm10
Supplementary files format and content: Cellranger output files: Tab-separated values and matrix files
Supplementary files format and content: For ADT data: feature_reference.csv files

molecule: total RNA

organism: Mus musculus

source_name: Lymph node

extract_protocol:
The droplet-based approach of 10X Genomics platform was used to perform scRNA-seq. For each scRNA-seq sample, cell suspension from three individual mice were pooled and sorted in PBS containing 0,04%BSA.
10 000 cells were loaded on a Chromium Controller using the Chromium Next GEM Single Cell 3 Reagent Kits v3.1 according to manufacturer instructions.

treatment_protocol: In this project, we have worked in the KP (KRASG12D; p53-/-) model of murine lung cancer. 14 days after intravenous injection of the KP cell line, CD8+ T cells or CD11c+MHCII+ cells were stained with CITE-Seq antibodies then sorted by flow cytometry from pooled lungs or lymph nodes of 3 C57BL/6J mice before scRNA-seq analysis.Single cells were isolated using a 10X Chromium droplet-based approach and sequenced.

value: Lymph node

tag: cell line

value: KP

tag: cell type

value: CD8+ T cells

tag: strain

tag: genotype

value: WT

tag: age

value: 8-12 weeks

tag: library type

value: RNA


title: Nasal epithelial brushing, PARDS subject ARDS_021

type: genomic

city: Little Rock

first: James

middle: Garrett

last: Williams

state: AR

address: 13 Children's Way

department: Pediatrics

country: USA

institute: University of Arkansas for Medical Sciences

hyb_protocol: Bisulfite-converted, amplified DNA was enzymatically fragmented and hybridized to the Illumina Infinium MethylationEPIC v2.0 BeadChip, followed by single-base extension and staining, per the Illumina Infinium HD Methylation Assay protocol (CCHMC DNA Sequencing and Genotyping Core).

scan_protocol: BeadChips were imaged on the Illumina iScan system.

data_processing:
IDAT files were processed in R (v4.5.0) with the sesame package. openSesame was run with prep='QCDPB' (quality masking, channel/dye-bias correction, noob background correction, and pOOBAH detection-p masking) on platform EPICv2 using a pinned local manifest; beta values were computed with getBetas. Probes flagged by the EPICv2 KYCG mask (cross-reactive and SNP-affected) and probes with detection p-value > 0.05 were removed. Replicate EPICv2 probes were collapsed to one value per CpG by averaging (limma::avereps on the base CpG identifier). M-values were computed as log2(beta/(1-beta)). Cell-type composition was estimated with EpiDISH, and M-values were adjusted for composition (limma::removeBatchEffect) for downstream analyses. The submitted processed matrix contains normalized beta values per CpG per sample.
[processed value definition] Beta value = methylated signal / (methylated + unmethylated signal), ranging from 0 (unmethylated) to 1 (fully methylated), after noob/dye-bias correction and pOOBAH detection-p masking (sesame getBetas). NA indicates a probe that failed detection in that sample.

label: Cy3 and Cy5

molecule: genomic DNA

organism: Homo sapiens

source_name: nasal epithelium

label_protocol: Standard Illumina Infinium HD Methylation Assay. Genomic DNA was bisulfite-converted and whole-genome amplified per the manufacturer's protocol.

extract_protocol: Nasal brushes were thawed on ice in 1:100 beta-mercaptoethanol to reduce mucus disulfide bonds, passed through a QIAshredder (Qiagen), and treated with DNase. Genomic DNA was extracted with the AllPrep DNA/RNA Mini Kit (Qiagen) and quantified on a Qubit Flex fluorometer (Thermo Fisher Scientific).

value: nasal epithelium

tag: condition

value: PARDS


title: CHI085 Silent Het

city: Memphis

first: Stanislav

last: Zakharenko

state: Tennessee

address: 262 Danny Thomas Pl, M2426

department: Developmental Neurobiology

institute: St. Jude Children's Research Hospital

description: Library name: CHI085

library_source: transcriptomic

data_processing:
Adapter sequences were removed by TrimGalore (v0.6.3).
STAR (v2.7.9a) was then used for alignment to the mouse reference genome (GRCm38) in a splice-site aware manner based on Gencode M22 primary assembly annotations.
Gene level quantifications were derived using RSEM (v1.3.3).
Assembly: Mouse mm10
Supplementary files format and content: Processed data contains raw counts per gene for each sample.

molecule: polyA RNA

source_name: dorsal striatum

extract_protocol:
Cell contents were extracted using the recording electrode following patch-clamp recordings. Contents were directly lysed in buffer from the SMART-Seq mRNA Single Cell LP kit (Takara, 634787).
Libraries were constructed from single cells using the SMART-Seq mRNA Single Cell LP kit (Takara, 634787).

value: dorsal striatum

tag: group

value: Silent

value: cholinergic interneuron

value: Het

tag: Sex

value: male

tag: batch

tag: date ephys

tag: dob

tag: age days

tag: electrophysiologist

tag: mouse id

tag: rt lot

tag: date lp

tag: lp lot

tag: index lot

tag: date seq

tag: seq srm_sample_id


title: CHI084 Active Het

description: Library name: CHI084

tag: tissue

value: Active

tag: cell type

tag: genotype


title: PFUnDA-2

city: Beijing

country: China

description:
Library name: sample8
PFUnDA-2

value: liver

tag: cell line

value: hepatic organoids

tag: treatment

value: 100  μM  PFUnDA


title: BMAL2_SEV_3

city: St Louis

first: Carolina

middle: B

last: Lopez

state: MO

address: 425 S Euclid Ave

institute: Washington University in St. Louis

description: Library name: BMAL2_SEV_3

data_processing:
Quality of the fastq files was assessed using FastQC v0.12.1. Reads were then quality filtered using fastp v0.24.0 with poly-X tail trimming, 3' quality-based tail trimming, a minimum Phred quality score of 15, and a minimum length requirement of 50 bp. Quality-filtered reads were aligned to the reference genome using STAR aligner v2.7.11 with non-canonical splice junction removal and output of unmapped reads, followed by coordinate sorting using samtools v1.22.1. PCR and optical duplicates were removed using UMI-based deduplication with UMIcollapse v1.1.0. Alignment quality metrics, strand specificity, and read distribution across genomic features were assessed using RSeQC v5.0.4 and Qualimap v2.3, with results aggregated into a comprehensive quality control report using MultiQC v1.32. Gene-level expression quantification was performed using featureCounts (subread package v2.1.1) with strand-specific counting, multi-mapping read fractional assignment, exons and three prime UTR as the feature identifiers,  and grouped by gene_id. Final gene counts were annotated with gene biotype and  other metadata extracted from the reference GTF file.  Sample-sample correlations  for sample-sample heatmap and PCA were calculated on normalized counts  (TMM, trimmed mean of M-values) using Pearson correlation. Differential expression  was done with edgeR v4.0.16 using standard practice including filtering for  low-expressed genes with edgeR::filterByExprwith default values. Functional  enrichment, when available, is performed using gene set enrichment analysis with  gseapy v0.12 using the MSigDB Hallmark gene set.
Assembly: GRCh38
Supplementary files format and content: fastq sequencing files
Supplementary files format and content: csv gene expression tables
Supplementary files format and content: csv gene set enrichment tables

source_name: BMAL2 A549

growth_protocol: A549 human lung epithelial cells (ATCC CCL-185) were maintained in Dulbecco’s Modified Eagle Medium (DMEM; Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 1 mM sodium pyruvate, and 50 μg/mL gentamicin at 37°C in a humidified incubator containing 5% CO2.

extract_protocol:
RNA was extracted by Kingfisher (Thermofisher) with MagMax Mirvana Total RNA isolation kit (Thermofisher, A27828).
Poly-A mRNA capture and reverse transcription for cDNA synthesis: oligo-dT primer to capture just the mRNA (contains sample barcode, UMI, and Read 1 sequences). Second strand synthesis and tagmentation: double stranded-cDNA is generated. Tagmentation creates fragments and incorporates Read 2 sequence. Illumina library amplification: adds unique dual indices (UDI = i5, i7) and P5/P7 sequences.

treatment_protocol: Cells were infected with Sendai Virus (MOI 1.5) for 24 hours

value: BMAL2 A549

value: lung epithelial cells

value: SeV infected


title: ChIPseq, H3K27ac, aux, rep2

city: Tokyo

first: Shoin

last: Tei

address: 1-1-1 Yayoi, Bunkyo-ku

department: Institute for Quantitative Biosciences

country: Japan

institute: The University of Tokyo

library_source: genomic

data_processing:
Sequenced reads were aligned to hg38 and mm10 using bowtie (version 1.2.3) with “-n2-m1” parameters. The removal of redundant reads and the normalization of the read number for each sample were performed with parse2wig (version 3.7.2). For peak-calling, we used drompa_peakcall (version 3.7.2) in “PC_SHARP” mode, and for visualization, we used drompa+ PC_SHARP (version 1.17.2) and deeptools (version 3.5.1.)
Assembly: hg38
Supplementary files format and content: BigWig files generated by parse2wig (version 3.7.2)

growth_protocol: HCT116 cells were cultured in McCoy’s 5A medium containing 10% FBS, penicillin, and streptomycin solution (Wako) at 37 C with 5% CO2.

extract_protocol:
HCT116 and C2C12 cells were crosslinked with 1% formaldehyde for 10 min, quenched with 125 mM glycine for 5 min, and washed with PBS. After collection by centrifugation, the fixed cells were frozen and stored at -80 C. The thawed cells resuspended with LB1 Buffer (50 mM HEPES-KOH (pH 7.5), 140 mM NaCl, 1 mM EDTA, 10 % Glycerol, 0.5 % NP-40, 0.25 % Triton-X, 1 mM PMSF, 10 mM DTT, 1x cOmplete EDTA-free (Roche)) and incubated on ice for 10 min. After collection by centrifugation, the pellet was resuspended with LB2 Buffer (20 mM Tris-HCl (pH7.5), 200 mM NaCl, 1 mM EDTA, 1 mM PMSF, 1x cOmplete EDTA-free) and incubated on ice for 10 min. After centrifugation, the pellet was resuspended with LB3 Buffer (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 % Triton-X, 0.1 % Na-Deoxycholate, 0.1 % SDS, 1x cOmplete EDTA-free) and incubated on ice for 10 min. Then, we repeated the centrifugation and resuspension with LB3 Buffer. For shearing chromatin, sonication was performed for 12 sec 6 times with amplitude setting at 17 % of the maximum amplitude using Sonifier 250D. Between each cycle, chromatin was collected by centrifugation. After the step of sonication, cell debris was removed by centrifugation at 19000 g for 15 min. 50 μl of Dynabeads Protein A or Dynabeads Protein G (Invitrogen) was washed with 5 mg/ml BSA/PBS twice and resuspended with BSA/PBS. 4 μg antibody was added to the beads, and the mixture was rotated for 3 h at 4 C. The beads was washed with BSA/PBS twice and with LB3 Buffer once and resuspended with LB3 Buffer. Chromatin from HCT116 and from C2C12 cells were mixed and incubated with antibody beads for 14 h at 4 C. The beads was washed with LB3 Buffer once, with LB3 High Buffer ( 20 mM Tris-HCl (pH 7.5), 500 mM NaCl, 1 mM EDTA, 1 % Triton-X, 0.1 % Na-Deoxycholate, 0.1 % SDS) twice, with RIPA wash Buffer (50 mM HEPES-KOH (pH 7.4), 0.25 M LiCl, 1 mM EDTA, 0.5 % Na-Deoxycholate, 1 % NP-40) 3 times and with TE50 Buffer (50 mM Tris-HCl (pH 7.5), 10 mM EDTA) once. Chromatin bound to beads was eluted with Elution Buffer (50 mM Tris-HCl (pH 7.5), 10 mM EDTA, 1 % SDS). The eluates were incubated at 65 °C for 6 h to reverse crosslinks and then treated with RNaseA and then with proteinase K. The samples were purified with QIAquick PCR purification kit (QIAGEN).  Methods Mol Biol, 2017. 1515: p. 257-271).
The library for sequencing was prepared with NEBNext DNA Library Prep Master Mix Set for Illumina and NEBNext Multiplex Oligos for Illumina (New England Biolabs).

treatment_protocol: Treatment with 0.5 mM indole-3-acetic acid (IAA, SIGMA) for 3h was performed to deplete Rad21.

tag: chip antibody

value: Colorectal carcinoma

value: Auxin


title: Lung, CD8, KP, WT, RNA

description:
Library name: Lung_CD8_KP_WT_RNA
10x genomics
RNA

source_name: Lungs

value: Lungs

value: KP

value: CD8+ T cells

tag: strain

value: WT

tag: age

value: 8-12 weeks

tag: library type

value: RNA


title: CHI083 Active Het

description: Library name: CHI083

value: dorsal striatum

tag: group

value: cholinergic interneuron

value: Het

tag: Sex

tag: batch

tag: date ephys

tag: dob

tag: age days

tag: electrophysiologist


title: PFUnDA-1

description:
Library name: sample7
PFUnDA-1

value: liver

value: Liver

value: 100 μM PFUnDA


title: CD103pos_Tact, rep2

description:
Library name: CD103pos_Tact_2
Column name in tablecounts_normalized_LN_T_cells.csv: CD103posTactKPDay14_2

data_processing:
Analysis was performed following the conventional DESeq2 package instructions.
Assembly: mm10
Supplementary files format and content: For RNA-Seq samples: csv files include normalized counts for each sample

extract_protocol:
After tissue processing and cell count, cell suspensions were washed in cold FACS buffer (PBS + 3% BSA + 2mM EDTA), Fc-receptors were blocked ,15min at 4°C, using purified anti-CD16/32 (2.4G2) and, then, stained with fluorophore conjugated antibodies for 30 minutes at 4°C. CD8+T cell subsets from tumor-bearing lungs and tdLNs were isolated using an Aria III (BD) and a MELODY (BD) cell sorter, respectively, and directly collected on lysing TCL buffer (QIAGEN) containing 0.1% of beta-mercaptoethanol before storage at -80°C.
RNA were extracted and isolated using the Single Cell RNA purification kit (Norgen, Cat#51800) according to the manufacturer’s instructions. After extraction, total RNA was analyzed using Agilent RNA 6000 Pico Kit on the Agilent 2100 Bioanalyzer System. RNA quality was estimated based on capillary electrophoresis profiles using the RNA Integrity Number (RIN). RNA sequencing libraries were prepared using the SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian (Takara). The input quantity of total RNA was comprised between 1 and 22ng. This protocol includes a first step of RNA fragmentation, using a proprietary fragmentation mix at 94°C. The time of incubation was set up for each sample, based on the RNA quality, and according to the manufacturer’s recommendations. After fragmentation, indexed cDNA synthesis was performed. Then the ribodepletion step was performed, using probes specific to mammalian rRNA. PCR amplification was finally achieved to amplify the indexed cDNA libraries, with a number of cycles set up according to the input quantity of tRNA. Library quantification and quality assessment was performed using Qubit fluorometric assay (Invitrogen) with dsDNA HS (High Sensitivity) Assay Kit and LabChip GX Touch using a High Sensitivity DNA chip (Perkin Elmer). Libraries were then equimolarly pooled and quantified by qPCR using the KAPA library quantification kit (Roche). Sequencing was carried out on the NovaSeq 6000 (Illumina), targeting between 10 and 15M reads per sample and using paired-end 2 x 100 bp.

treatment_protocol: In this project, we have worked in the KP (KRASG12D; p53-/-) model of murine lung cancer. Briefly, the KP cell line was injected intravenously in C57BL/6J mice and CD8+ T cell and dendritic cell populations were assessed at day 14 post-injection. Two bulk RNA-Seq datasets were generated: - CD8+ T cells populations from individual lungs or lymph nodes of 3-4 KP tumor-bearing mice were FACS-sorted and bulk-RNA sequencing was performed. - CTV-loaded naïve OT-I cells cultured for 2 days with flow cytometry-sorted migratory DC1 or migratory DC2 from tumor-draining lymph node of mice bearing KP-OVA tumors. CD103+ and CD103- divided OT1 activated by DC1 or DC2 were FACS-sorted and bulk-RNA sequencing was performed.

value: Lymph node

value: CD103pos_activated_CD8+ T cells


title: ChIPseq, H3K27ac, ctrl, rep2

value: DMSO


title: CHI082 Silent WT

description: Library name: CHI082

value: Silent

value: male

tag: mouse id

tag: rt lot

tag: date lp

tag: lp lot

tag: index lot

tag: date seq

tag: seq srm_sample_id


title: CD103pos_Tact, rep1

description:
Library name: CD103pos_Tact_1
Column name in tablecounts_normalized_LN_T_cells.csv: CD103posTactKPDay14_1

tag: tissue

tag: cell line

tag: cell type

tag: strain

tag: genotype


title: PFOA-3

description:
Library name: sample6
PFOA-3

value: hepatic organoids

tag: treatment

value: 100 μM  PFOA


title: ChIPseq, Input, H3K27ac, aux, rep1

tag: chip antibody

value: Colorectal carcinoma

value: Auxin


title: CHI081 Silent WT

description: Library name: CHI081

tag: group

value: WT

tag: Sex

tag: batch

tag: date ephys

tag: dob


title: CD103neg_Tact, rep3

description:
Library name: CD103neg_Tact_3
Column name in tablecounts_normalized_LN_T_cells.csv: CD103negTactKPDay14_3

value: KP

value: CD103neg_activated_CD8+ T cells

tag: age

value: 8-12 weeks


title: ChIPseq, Input, H3K27ac, ctrl, rep1


title: PFOA-2

description:
Library name: sample5
PFOA-2


title: CHI078 Silent WT

description: Library name: CHI078

value: dorsal striatum

value: cholinergic interneuron

tag: age days

tag: electrophysiologist

tag: mouse id

tag: rt lot

tag: date lp

tag: lp lot


title: PFOA-1

description:
Library name: sample4
PFOA-1

tag: tissue

value: liver

tag: cell line

tag: cell type

value: Liver


title: ChIPseq, H3K27ac, aux, rep1

tag: genotype

tag: treatment


title: CHI077 Active WT

description: Library name: CHI077

tag: group

value: Active

tag: index lot

tag: date seq

tag: seq srm_sample_id


title: CD103neg_Tact, rep1

description:
Library name: CD103neg_Tact_1
Column name in tablecounts_normalized_LN_T_cells.csv: CD103negTactKPDay14_1

value: Lymph node

tag: strain


title: ChIPseq, Input, H3K27ac, ctrl, rep2

tag: chip antibody

value: DMSO


title: DMSO-3.

description:
Library name: sample3
DMSO-3

value: 0.1% DMSO


title: Trm_Tim3_pos, rep3

description:
Library name: Trm_Tim3_pos_3
Column name in tablecounts_normalized_lung_T_cells.csv: TrmTim3pos_3

value: Lungs

value: Trm_Tim3pos_CD8+ T cells

tag: age


title: CHI076 Silent WT

description: Library name: CHI076

value: Silent

tag: Sex

value: male

tag: batch

tag: date ephys

tag: dob

tag: age days

tag: electrophysiologist


title: ChIPseq, Ser2P, aux

tag: cell line

value: Colorectal carcinoma

value: Auxin


title: CHI075 Active WT

description: Library name: CHI075

tag: tissue

tag: cell type

value: WT

tag: mouse id

tag: rt lot

tag: date lp

tag: lp lot


title: Trm_Tim3_pos, rep2

description:
Library name: Trm_Tim3_pos_2
Column name in tablecounts_normalized_lung_T_cells.csv: TrmTim3pos_2

value: KP

tag: genotype


title: DMSO-1.

description:
Library name: sample1
DMSO-1

value: hepatic organoids

tag: treatment


title: ChIPseq, Ser2P, ctrl

tag: chip antibody


title: Trm_Tim3_pos, rep1

description:
Library name: Trm_Tim3_pos_1
Column name in tablecounts_normalized_lung_T_cells.csv: TrmTim3pos_1

tag: strain

tag: age

value: 8-12 weeks


title: ChIPseq, Input, RPB1, aux, flav, 15

value: Flavopiridol washout 15min


title: CHI073 Active WT

description: Library name: CHI073

value: dorsal striatum

tag: group

value: cholinergic interneuron

tag: Sex

tag: batch

tag: index lot

tag: date seq

tag: seq srm_sample_id


title: Trm_Tim3_neg, rep3

description:
Library name: Trm_Tim3_neg_3
Column name in tablecounts_normalized_lung_T_cells.csv: TrmTim3neg_3

tag: tissue

tag: cell line

value: Trm_Tim3neg_CD8+ T cells


title: CHI072 Active WT

description: Library name: CHI072

value: Active

tag: cell type

tag: date ephys

tag: dob

tag: age days

tag: electrophysiologist

tag: mouse id


title: Jur_KO_CD3_CT2

description:
Library name: sample23
Jur_KO_CD3_CT2

source_name: cell line

growth_protocol: Human Jurkat T cells were purchased from American Type Culture Collection (ATCC). Jurkat T cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 (Thermo Scientific) complete medium with 10% (vol/vol) fetal bovine serum (FBS, Thermo Scientific, Cat: 10270106, New York, NY, USA) and 1%(vol/vol) penicillin/streptomycin (P/S,Thermo Scientific, Cat: 15140122, New York, NY, USA).

treatment_protocol: The PMA and Ionomycin were dissolved in Dimethyl sulfoxide (DMSO) to prepare a stock solution of 320 μM and 5.2 mM, which were stored at −20 ℃. The stock solution was further diluted 2000−fold to prepare a working solution, which was subsequently treated with Jurkat T cells. The Jurkat cells were stimulated with PMA/ionomycin (0.16/2.6 mM) for 24 h, followed by RNA sequencing. The anti-human CD3 activation antibody and anti-human CD28 activation antibody were stored at 4℃. The Jurkat cells were stimulated with anti-CD3/CD28 mAbs (5/2 μg/mL) for 24 h, followed by RNA sequencing.

value: cell line

value: Human Jurkat T cell

tag: genotype

tag: treatment


title: ChIPseq, Input, RPB1, ctrl, flav, 15

tag: chip antibody

value: Flavopiridol washout 15min, Auxin


title: Trm_Tim3_neg, rep2

description:
Library name: Trm_Tim3_neg_2
Column name in tablecounts_normalized_lung_T_cells.csv: TrmTim3neg_2

value: Lungs

tag: strain

tag: age


title: ChIPseq, RPB1, aux, flav, 15


title: CHI071 Silent WT

description: Library name: CHI071

tag: group

value: Silent

tag: Sex

tag: batch

tag: rt lot

tag: date lp

tag: lp lot


title: Jur_KO_CD3_ACT3

description:
Library name: sample21
Jur_KO_CD3_ACT3

tag: tissue

tag: cell line

tag: cell type


title: ChIPseq, RPB1, ctrl, flav, 15


title: CHI070 Silent WT

description: Library name: CHI070

value: male

tag: date ephys

tag: dob

tag: age days

tag: index lot

tag: date seq

tag: seq srm_sample_id


title: ChIPseq, Input, RPB1, aux, flav, 10

tag: chip antibody

value: Colorectal carcinoma

tag: genotype

tag: treatment

value: Flavopiridol washout 10min


title: Jur_KO_CD3_ACT2

description:
Library name: sample20
Jur_KO_CD3_ACT2


title: Not_Trm_PD1_pos_Tim3_pos, rep2

description:
Library name: Not_Trm_PD1_pos_Tim3_pos_2
Column name in tablecounts_normalized_lung_T_cells.csv: NotTrmPD1posTim3pos_2

value: Not_Trm_PD1pos_Tim3pos_CD8+ T cells

tag: strain

value: WT

tag: age


title: Jur_KO_CD3_ACT1

description:
Library name: sample19
Jur_KO_CD3_ACT1


title: ChIPseq, Input, RPB1, ctrl, flav, 10

value: Flavopiridol washout 10min, Auxin


title: Not_Trm_PD1_pos_Tim3_pos, rep1

description:
Library name: Not_Trm_PD1_pos_Tim3_pos_1
Column name in tablecounts_normalized_lung_T_cells.csv: NotTrmPD1posTim3pos_1

value: KP

value: 8-12 weeks


title: Jur_WT_CD3_CT3

description:
Library name: sample18
Jur_WT_CD3_CT3


title: CHI068 Active WT

description: Library name: CHI068

value: dorsal striatum

tag: group

tag: Sex

tag: batch

tag: electrophysiologist

tag: mouse id

tag: rt lot


title: CHI067 Active WT

description: Library name: CHI067

tag: tissue

tag: cell type

value: cholinergic interneuron

tag: date ephys

tag: date lp

tag: lp lot


title: Jur_WT_CD3_CT2

description:
Library name: sample17
Jur_WT_CD3_CT2

tag: cell line


title: ChIPseq, RPB1, ctrl, flav, 10

tag: chip antibody


title: Not_Trm_PD1_pos_Tim3_neg, rep3

description:
Library name: Not_Trm_PD1_pos_Tim3_neg_3
Column name in tablecounts_normalized_lung_T_cells.csv: NotTrmPD1posTim3neg_3

value: Not_Trm_PD1pos_Tim3neg_CD8+ T cells

tag: genotype


title: V2 Pool 2 DNA Replicate 3

city: Boston

first: Jeerthi

last: Kannan

state: MA

address: 401 Park Dr

department: Nephrology

institute: Boston Children's Hospital

description:
Library name: V2_Pool2_DNA3
dna.r3 column in mpra_qtigc_pgl4v2_pool2.txt

library_source: other

data_processing:
For oligo sequencing, we preprocessed the raw  data using the NGMerge tool (Gaspar, BMC BIoinformatics, 2018)  to combine paired reads and remove flanking adaptor sequences. We then aligned the merged reads to the designed oligo sequences using the Bowtie2 in the end-to-end mode, with the --very-sensitive option. Next, we identified perfectly aligned reads using the optional tag AS=0. We also extracted mismatch information from the XM tags and INDEL information from the XG tags.
For barcode sequencing (both RNA and DNA), we combined the paired-end reads using the NGMerge tool and used only the 13 bp barcode sequences. We used only perfect matches of the designed barcodes for both DNA and mRNA quantification.
Supplementary files format and content: tab-separated value text files

molecule: other

organism: Escherichia coli

growth_protocol: Mouse cardiomyocyte HL-1 cells were cultured in Claycomb medium (Sigma)

extract_protocol:
The two 200-mer oligo pools (Twist Bioscience) were first cloned into pLS-mP vector backbone, before sub-cloning into pGL4.23 (Promega) vector backbone. pLS-mP was digested with SbfI and EcoRI (New England Biolabs) to release the minimal promoter-driven eGFP gene. Oligo pools were PCR amplified using primers that are specific to the 5’ and 3’ flanking sequences and introduce overhangs complementary to the SbfI and EcoRI digested pLS-mP vector backbone. Post gel extraction cleanup (Zymo Research), amplified products were cloned using HiFi DNA Assembly master mix (New England Biolabs). The overhangs complementary to the vector backbone were designed to disrupt the original SbfI and EcoRI sites in the vector post-assembly. The cloning reaction was transformed into electrocompetent Stable cells, and transformants (>5 million/pool)  were pooled, grown overnight and maxi-prepped (Thermo Fisher Scientific) to generate the pre-reporter MPRA plasmid libraries for each of the two oligo pools (Pre-reporter/pLS-mP/Stable). These plasmid libraries were then used to generate the oligo sequencing Illumina libraries and sub-cloning of oligos into pGL4.23 vector backbone. We generated these cloned oligo libraries for potential use in future for lentiviral delivery-based MPRA.
For sub-cloning of oligos into pGL4.23, pGL4.23 DNA was first digested with KpnI and XbaI (New England Biolabs) to release the minimal promoter-driven firefly luciferase gene and generate the vector backbone for cloning of MPRA oligos. The cloned oligos in the two pre-reporter MPRA plasmid libraries in pLS-mP backbone were PCR amplified using primers that are specific to the 5’ and 3’ flanking sequences and introduce overhangs complementary to the KpnI and XbaI digested pGL4.23 vector backbone. Post gel extraction cleanup (Zymo Research), amplified products were cloned using HiFi DNA Assembly master mix (New England Biolabs). The cloning reaction was transformed into electrocompetent Stable and DH10β cells, and transformants (>5 million/pool) were pooled, grown overnight and maxi-prepped (Thermo Fisher Scientific) to generate two versions, V1 and V2, of the pre-reporter MPRA plasmid libraries for each of the two oligo pools (V1: Pre-reporter/pGL4.23/Stable; V2: Pre-reporter/pGL4.23/DH10β). Two different E. coli host cells were used for library propagation to maximize representation and recovery of test elements. These pre-reporter plasmid libraries were digested with SbfI and EcoRI (New England Biolabs), sites for which are present only in the deigned oligos, and ligated with the minimal promoter-driven eGFP reporter cassette from pLS-mP, released by SbfI and EcoRI digestion. Like above, the cloning reaction was transformed into electrocompetent Stable and DH10β cells, and transformants (>5 million/pool) were pooled, grown overnight and maxi-prepped (Thermo Fisher Scientific) to generate two versions, V1 and V2, the post-reporter MPRA plasmid libraries for each of the two oligo pools (V1: Post-reporter/pGL4.23/Stable; V2: Post-reporter/pGL4.23/DH10β).
Quality of the synthesized oligos and their representation was assessed by high throughput sequencing. For each of the two oligo pools, oligos cloned in the pre-reporter MPRA plasmid libraries (Pre-reporter/pLS-mP/Stable) were amplified using custom primers that include P5 and P7 Illumina adapter sequences to generate custom amplicon single-indexed oligo sequencing libraries. These two libraries were pooled and sequenced on Illumina MiSeq platform (2×170 + 8 base index) using custom sequencing primers to cover the entire oligo except for the 5’ and 3’ constant flanking sequences.
The post-reporter MPRA plasmid libraries (V1 and V2) were transfected using FuGENE HD Transfection Reagent (Promega) into HL-1 cells, 24h post seeding of 5 million cells in 10 cm dishes, in 10 parallel replicates per pool to ensure robustness and reproducibility. To preserve library complexity, the number of cells at the time of transfection ensures that at least 50× more cells get transfected as compared to the number of unique oligos after adjusting for ~40% transfection efficiency. Forty-eight hours post- transfection cells were harvested, and total RNA was isolated using QIAshredder (Qiagen) and RNeasy Mini Kit (Qiagen). All RNA isolated was used to isolate mRNA using oligo-dT based Magnetic mRNA Isolation Kit (New England Biolabs), which was then treated with TURBO DNase (Thermo Fisher Scientific) to remove contaminating DNA. 500 ng of mRNA from each replicate was reverse transcribed with SuperScript III First-Strand Synthesis System (Thermo Fisher Scientific) in two parallel reactions using a primer downstream of the barcode, which contained a sample index and P7 Illumina adapter sequence. The two cDNA preparations for each replicate transfection were combined, and used as a template in PCR to generate the cDNA (RNA) -based MPRA barcode sequencing library with the same reverse primer and a forward primer complementary to the 3’ end of eGFP with a P5 Illumina adapter sequence. Similarly, the post-reporter MPRA plasmid libraries (V1 and V2) for each oligo pool were used as templates in PCR to generate the input plasmid (DNA) -based MPRA barcode sequencing libraries in three replicates. In total, we generated 26 (3 replicates for input plasmid DNA and 10 transfection replicates for RNA for each of the two oligo pools) custom amplicon single-indexed barcode libraries for V1, which were pooled and sequenced on the Illumina NextSeq 550 platform (2×26 + 8 base index; high-output kit) using custom sequencing primers. The same was repeated for V2.


title: ChIPseq, H3K27ac, ctrl, rep1

tag: treatment

value: DMSO


title: Not_Trm_PD1_pos_Tim3_neg, rep2

description:
Library name: Not_Trm_PD1_pos_Tim3_neg_2
Column name in tablecounts_normalized_lung_T_cells.csv: NotTrmPD1posTim3neg_2

tag: strain


title: ChIPseq, Input, RPB1, aux, flav, 0

value: Flavopiridol,Auxin


title: Jur_KO_CD3_CT3

description:
Library name: sample24
Jur_KO_CD3_CT3

value: cell line

value: Human Jurkat T cell


title: CHI074 Active WT

description: Library name: CHI074

value: Active

tag: dob

tag: age days

tag: index lot

tag: date seq

tag: seq srm_sample_id


title: V2 Pool 2 DNA Replicate 2

description:
Library name: V2_Pool2_DNA2
dna.r2 column in mpra_qtigc_pgl4v2_pool2.txt


title: Jur_WT_CD3_ACT3

description:
Library name: sample15
Jur_WT_CD3_ACT3


title: Jur_WT_CD3_ACT2

description:
Library name: sample14
Jur_WT_CD3_ACT2


title: Not_Trm_PD1neg, rep3

description:
Library name: Not_Trm_PD1neg_3
Column name in tablecounts_normalized_lung_T_cells.csv: NotTrmPD1neg_3

value: Lungs

value: Not_Trm_PD1neg_CD8+ T cells

tag: age


title: V2 Pool 2 DNA Replicate 1

description:
Library name: V2_Pool2_DNA1
dna.r1 column in mpra_qtigc_pgl4v2_pool2.txt


title: V2 Pool 1 DNA Replicate 3

description:
Library name: V2_Pool1_DNA3
dna.r3 column in mpra_qtigc_pgl4v2_pool1.txt


title: V2 Pool 1 DNA Replicate 2

description:
Library name: V2_Pool1_DNA2
dna.r2 column in mpra_qtigc_pgl4v2_pool1.txt


title: Not_Trm_PD1neg, rep1

description:
Library name: Not_Trm_PD1neg_1
Column name in tablecounts_normalized_lung_T_cells.csv: NotTrmPD1neg_1


title: Jur_KO_PMA_CT3

description:
Library name: sample12
Jur_KO_PMA_CT3


title: ChIPseq, Input, RPB1, aux, rep2

value: Colorectal carcinoma

value: Auxin


title: ChIPseq, Input, RPB1, ctrl, flav, 0

value: Flavopiridol


title: V2 Pool 1 DNA Replicate 1

description:
Library name: V2_Pool1_DNA1
dna.r1 column in mpra_qtigc_pgl4v2_pool1.txt


title: ChIPseq, RPB1, ctrl, flav, 0


title: Not_Trm_PD1neg, rep2

description:
Library name: Not_Trm_PD1neg_2
Column name in tablecounts_normalized_lung_T_cells.csv: NotTrmPD1neg_2

value: WT


title: Divided_OT1_CD103pos, DC2act, rep4

description:
Library name: Divided_CD103pos_OT1_DC2act_4
Column name in tablecounts_normalized_ex_vivo_OT1.csv: DC2_CD103pos_4

value: Lymph node

value: KP-OVA

value: CD103pos_OT1 CD8+ T cells


title: Jur_WT_CD3_ACT1

description:
Library name: sample13
Jur_WT_CD3_ACT1


title: Jur_KO_PMA_CT2

description:
Library name: sample11
Jur_KO_PMA_CT2


title: Divided_OT1_CD103pos, DC2act, rep3

description:
Library name: Divided_CD103pos_OT1_DC2act_3
Column name in tablecounts_normalized_ex_vivo_OT1.csv: DC2_CD103pos_3

value: 8-12 weeks


title: V1 Pool 2 DNA Replicate 3

description:
Library name: V1_Pool2_DNA3
dna.r3 column in mpra_qtigc_pgl4v1_pool2.txt

source_name: Stable

value: Stable


title: ChIPseq, Input, RPB1, ctrl, rep2


title: Jur_KO_PMA_CT1

description:
Library name: sample10
Jur_KO_PMA_CT1


title: CD103neg_Tact, rep2

description:
Library name: CD103neg_Tact_2
Column name in tablecounts_normalized_LN_T_cells.csv: CD103negTactKPDay14_2

value: KP

value: CD103neg_activated_CD8+ T cells


title: ChIPseq, RPB1, aux, rep2


title: Jur_KO_PMA_ACT3

description:
Library name: sample9
Jur_KO_PMA_ACT3


title: V1 Pool 2 DNA Replicate 2

description:
Library name: V1_Pool2_DNA2
dna.r2 column in mpra_qtigc_pgl4v1_pool2.txt


title: Divided_OT1_CD103pos, DC2act, rep2

description:
Library name: Divided_CD103pos_OT1_DC2act_2
Column name in tablecounts_normalized_ex_vivo_OT1.csv: DC2_CD103pos_2


title: ChIPseq, RPB1, ctrl, rep2

value: DMSO


title: DMSO-2.

description:
Library name: sample2
DMSO-2

value: liver

value: hepatic organoids

value: Liver

value: 0.1% DMSO


title: V1 Pool 2 DNA Replicate 1

description:
Library name: V1_Pool2_DNA1
dna.r1 column in mpra_qtigc_pgl4v1_pool2.txt


title: Divided_OT1_CD103pos, DC2act, rep1

description:
Library name: Divided_CD103pos_OT1_DC2act_1
Column name in tablecounts_normalized_ex_vivo_OT1.csv: DC2_CD103pos_1


title: V1 Pool 1 DNA Replicate 3

description:
Library name: V1_Pool1_DNA3
dna.r3 column in mpra_qtigc_pgl4v1_pool1.txt


title: Jur_KO_PMA_ACT2

description:
Library name: sample8
Jur_KO_PMA_ACT2

value: cell line

value: Human Jurkat T cell


title: ChIPseq, Input, RPB1, aux, rep1

value: Colorectal carcinoma

value: Auxin


title: ChIPseq, Input, RPB1, control

description: Library name: ChIPseq_Input_RPB1_control

data_processing:
Sequenced reads were aligned to hg38 and mm10 using bowtie (version 1.2.3) with “-n2-m1” parameters. The removal of redundant reads and the normalization of the read number for each sample were performed with parse2wig (version 3.7.2). For peak-calling, we used drompa_peakcall (version 3.7.2) in “PC_SHARP” mode, and for visualization, we used drompa+ PC_SHARP (version 1.17.2) and deeptools (version 3.5.1.)
Assembly: hg38
Supplementary files format and content: BigWig

extract_protocol:
Mouse C2C12 cells were used as a spike-in control. HCT-116 and C2C12 cells were crosslinked with 1% formaldehyde for 10 min at room temperature, quenched with 125 mM glycine for 5 min, washed with PBS, pelleted by centrifugation, and stored at –80°C until use. For standard ChIP-seq, thawed cells were resuspended in 1 mL of LB1 Buffer [50 mM HEPES-KOH (pH 7.5), 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100, 1 mM PMSF, 10 mM DTT, 1x cOmplete EDTA-free (Roche)] and incubated on ice for 10 min. After centrifugation, the pellet was resuspended in 1 mL of LB2 Buffer [20 mM Tris-HCl (pH 7.5), 200 mM NaCl, 1 mM EDTA, 1 mM PMSF, 1x cOmplete EDTA-free] and incubated on ice for 10 min. Following another centrifugation, the pellet was resuspended in 1 mL of LB3 Buffer [20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% Na-Deoxycholate, 0.1% SDS, 1x cOmplete EDTA-free] and incubated on ice for 10 min. The centrifugation and resuspension steps with LB3 buffer were repeated. Chromatin was sonicated on Sonifier 250D (BRANSON) at 17% of amplitude for six cycles of 12 sec each. Between each cycle, chromatin was collected by centrifugation. After chromatin fragmentation, insoluble debris was removed by centrifugation. HCT-116 and C2C12 chromatin were combined at a 4:1 ratio. To prepare antibody-conjugated beads, 50 μL of Dynabeads Protein A/G (Invitrogen) were washed twice with PBS containing 5 mg/mL BSA and resuspended in the same buffer. After the addition of 4 μg of antibody, the mixture was rotated for 3 hours at 4°C. The beads were then washed twice with BSA/PBS and once with LB3 buffer, followed by resuspension in LB3 buffer. Chromatin mixture was rotated with the antibody-conjugated beads for 14 hours at 4°C. The beads were washed with LB3 Buffer once, twice with LB3 High Buffer [20 mM Tris-HCl (pH 7.5), 500 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% Na-Deoxycholate, 0.1% SDS], three times with RIPA wash Buffer [50 mM HEPES-KOH (pH 7.4), 0.25 M LiCl, 1 mM EDTA, 0.5% Na-Deoxycholate, 1% NP-40], and once with TE50 Buffer [50 mM Tris-HCl (pH 7.5), 10 mM EDTA]. Bead-bound DNA was eluted with Elution Buffer [50 mM Tris-HCl (pH 7.5), 10 mM EDTA, 1% SDS] and incubated at 65°C for 6 hours, followed by sequential treatment with RNase A and proteinase K. The samples were purified with QIAquick PCR purification kit (QIAGEN).
The library for sequencing was prepared with NEBNext DNA Library Prep Master Mix Set for Illumina and NEBNext Multiplex Oligos for Illumina (New England Biolabs).

treatment_protocol: Treatment with 0.5 mM indole-3-acetic acid (IAA, SIGMA) for 3h was performed to deplete RAD21. 100 μM 5,6-Dichlorobenzimidazole 1-β-D-Ribofuranoside (DRB; TCI) was added concurrently with IAA for 3 hours, or 1 μM flavopiridol (Santa Cruz) during the final 1 hour of the 3-hour IAA treatment.


title: CHI223 Active Het

description: Library name: CHI223

value: dorsal striatum

tag: group

value: Active

value: cholinergic interneuron

value: Het

tag: Sex

value: male

tag: batch

tag: electrophysiologist

tag: mouse id

tag: rt lot


title: Divided_OT1_CD103neg, DC2act, rep4

description:
Library name: Divided_CD103neg_OT1_DC2act_4
Column name in tablecounts_normalized_ex_vivo_OT1.csv: DC2_CD103neg_4

tag: tissue

value: Lymph node

tag: cell line

value: KP-OVA

tag: cell type

value: CD103neg_OT1 CD8+ T cells

value: WT

value: 8-12 weeks


title: Jur_KO_PMA_ACT1

description:
Library name: sample7
Jur_KO_PMA_ACT1

tag: genotype

tag: treatment


title: V1 Pool 1 DNA Replicate 2

description:
Library name: V1_Pool1_DNA2
dna.r2 column in mpra_qtigc_pgl4v1_pool1.txt

value: Stable


title: ChIPseq, Input, RPB1, ctrl, rep1

tag: chip antibody

value: DMSO


title: Divided_OT1_CD103neg, DC2act, rep3

description:
Library name: Divided_CD103neg_OT1_DC2act_3
Column name in tablecounts_normalized_ex_vivo_OT1.csv: DC2_CD103neg_3

tag: strain


title: Jur_WT_PMA_CT3

description:
Library name: sample6
Jur_WT_PMA_CT3


title: V1 Pool 1 DNA Replicate 1

description:
Library name: V1_Pool1_DNA1
dna.r1 column in mpra_qtigc_pgl4v1_pool1.txt


title: ChIPseq, RPB1, aux, rep1


title: Jur_KO_CD3_CT1

description:
Library name: sample22
Jur_KO_CD3_CT1


title: Divided_OT1_CD103neg, DC2act, rep2

description:
Library name: Divided_CD103neg_OT1_DC2act_2
Column name in tablecounts_normalized_ex_vivo_OT1.csv: DC2_CD103neg_2


title: Trm_Tim3_neg, rep1

description:
Library name: Trm_Tim3_neg_1
Column name in tablecounts_normalized_lung_T_cells.csv: TrmTim3neg_1

value: Lungs

value: KP

value: Trm_Tim3neg_CD8+ T cells


title: Jur_WT_PMA_CT2

description:
Library name: sample5
Jur_WT_PMA_CT2

value: cell line

value: Human Jurkat T cell


title: ChIPseq, RPB1, ctrl, rep1

value: Colorectal carcinoma


title: Divided_OT1_CD103neg, DC2act, rep1

description:
Library name: Divided_CD103neg_OT1_DC2act_1
Column name in tablecounts_normalized_ex_vivo_OT1.csv: DC2_CD103neg_1


title: stomach female high-dose exposure rep8

city: Durham

first: Weichun

last: Huang

state: NC

address: 109 TW Alexander Dr

department: OCSPP

institute: US EPA

description: Library name: S_48

data_processing:
RNA-seq data quality was assessed using FASTQC (v0.12.1) and summarized with MultiQC (v1.28)
PCR duplicates were removed by deduplicating sequencing reads with UMI-tools (v1.1.5)
Low-quality bases were trimmed using Cutadapt (v4.9) and Trim Galore (v0.6.10)
The cleaned reads were then aligned to the Rattus norvegicus genome assembly (mRatBN7.2; GCA_036323735.1) using STAR (v2.7.11b)
Quantification of transcript abundance was performed with Salmon (v1.10.3)
Assembly: the Rattus norvegicus genome assembly (mRatBN7.2; GCA_036323735.1
Supplementary files format and content: A tab-delimited matrix table with  raw read counts at the  transcrit level generated by salmon  (*.gene_counts.tsv )
Supplementary files format and content: A tab-delimited matrix table with  raw read counts at the  transcript level generated by salmon  (*.transcript_counts.tsv )

organism: Rattus norvegicus

source_name: stomach

growth_protocol: Rats were fed a standard rodent chow

extract_protocol:
A 50% (v/v) solution of Antifoam A (cat. no. A5633; Sigma, St. Louis, MO) was prepared in propylene glycol (cat. no. W294004; Sigma St. Louis, MO). The solution was vortexed prior to use. Homogenization buffer was prepared by adding 10 µl of β-mercaptoethanol (cat no. 0482, VWR, Radnor, PA) and 60 µl antifoam solution per milliliter of Buffer RLT Plus (Qiagen, Germantown, MD).
Prior to homogenization, large tissue samples were broken into smaller pieces using a mortar and pestle cooled with liquid nitrogen (LN2). Several of the resulting pieces (each approximately 3–5 mm) were then pulverized together to produce the final sample. For homogenization, 400 µl of buffer was added to a 3 ml, flat-bottom polystyrene tube (Globe Scientific, Mahwah, NJ). The tube was placed into a CoolRack CF15 (Corning Inc., Corning, NY) cooled with LN2. A cooled 30 µl plastic scoop was used to transfer pulverized tissue to the tube. The target mass of tissue was 50 µg, although the actual mass varied due to the need to work quickly and differences in the sizes of the pulverized samples.
The tube was placed on ice, and 600 µl of ice-cold homogenization buffer was added to the sample. The sample was homogenized using a model 985-370 TissueTearor rotor-stator homogenizer with a 7 mm probe (BioSpec Products, Bartlesville, OK) until no remnants of intact tissue were visible. If the homogenate appeared significantly darker than other samples, suggesting an excess of tissue, additional buffer was added until the color of the sample was similar to that of the other samples. Homogenates were stored at -80 °C until used for RNA isolation. The homogenizer was rinsed between samples within a treatment by running it at the highest speed for 30 s in three changes of deionized water. Between treatments, the rotor and stator were disassembled and cleaned with a polyester test tube brush before rinsing in water.
For RNA isolation, homogenized samples were removed from the freezer and placed on ice to thaw. The samples were vortexed, and 100 µl of the homogenate was transferred to the well of a 96-well round bottom plate (Caplugs Evergreen, Buffalo, NY). RNA was isolated using the MagMAX-96 Total RNA Isolation Kit (Fisher Scientific, Hampton, NH) according to the manufacturer’s protocol, except for the substitution of RLT Plus buffer for the lysis/binding solution as described above.
Isolated RNA was quantified on a Synergy HTX plate reader using a Take3 microvolume plate (Agilent, Santa Clara, CA). The integrity of the RNA was analyzed on an Agilent TapeStation using RNA Screen Tapes and reagents (Agilent, Santa Clara, CA). Concentrations of samples ranged from 75 to 470 ng/µl. RNA integrity numbers (RIN) ranged from 8.0 to 10.0; nearly 90% of samples had a RIN higher than 9.0.
Isolated RNA was next used to prepare RNA-Seq libraries. Samples were blocked so that about eight samples of each tissue and treatment combination were included in one of two plates. RNA was normalized to 25 ng/uL with nuclease-free water (Ambion, USA). 250 ng total RNA (10 uL of normalized RNA) was input into mRNA purification with magnetic beads using a PolyA Selection Module (Lexogen, USA). Purified mRNA was then used directly in CORALL mRNA-Seq V2 library preparation with set A unique dual indexes (UDIs). Each batch was checked by qPCR to determine the appropriate number of PCR cycles for library amplification with Add-On PCR V2 kit (Lexogen, USA), SYBR Green (ThermoFisher, USA), and a QuantStudio 6 Flex real-time PCR instrument (ThermoFisher, USA). After final PCR amplification, libraries were quantified with Quant-iT PicoGreen (ThermoFisher, USA). The distribution of library size was checked by TapeStation D1000 assay (Agilent, USA). The average library size was 508 – 517 bp. Samples below 1 ng/uL were re-amplified.

value: stomach

value: Sprague-Dawley

tag: developmental stage

value: adult

value: Female-High Dose/50mg/kg

value: Batch 1


title: Divided_OT1_CD103pos, DC1act, rep4

description:
Library name: Divided_CD103pos_OT1_DC1act_4
Column name in tablecounts_normalized_ex_vivo_OT1.csv: DC1_CD103pos_4

value: Lymph node

value: KP-OVA

value: CD103pos_OT1 CD8+ T cells


title: stomach female high-dose exposure rep7

description: Library name: S_47

value: WT


title: Neurons replicate 2 derived from iPSC replicate 3

city: Toronto

first: Shreejoy

last: Tripathy

address: 250 College Street

country: Canada

institute: Centre for Addiction and Mental Health

description: Library name: CN_3_2_150PM_CELL1

data_processing:
Basecalling used the PacBio basecaller v13.0.0 (default Revio parameters); HiFi (CCS) reads were generated with ccs v8.0.0, segmented into Kinnex S-reads with skera v1.0.99, demultiplexed/trimmed with lima v2.9.0, and refined to full-length non-chimeric (FLNC) reads using isoseq3 refine v3.4.0, after which reads were aligned to GRCh38 with minimap2 v2.26 using -ax splice:hq --secondary=no.
Assembly: GRCh38
Supplementary files format and content: tab-delimited file includes raw isoform counts for each sample

growth_protocol: PGPC17_11-CLYBL-TetO-NGN2 iPSCs were grown in 6-well plates coated with Matrigel (#354234, Corning) in mTeSR Plus media (#100-0276, STEMCELL) and passaged with ReLeSR (#100-0483, STEMCELL) for routine maintenance. All cells were kept at 37°C, 95% humidity and 5% CO2.

extract_protocol:
RNA was extracted using TRIzol™ Reagent according to the manufacturer's instructions
Full-length cDNA was first synthesized and amplified from total RNA using the Iso-Seq Express 2.0 Kit (PacBio). The resulting full-length cDNA was then used to prepare SMRTbell libraries with the Kinnex Full-Length RNA Kit (PacBio).

treatment_protocol: iPSCs were dissociated from maintenance culture plates with Accutase and seeded on Matrigel-coated 6-well plates at a density of 260,00 cells/cm² in custom complete neuron media with 10 μM ROCK inhibitor (Y-27632, #AB144494, Abcam) and 2 μM/mL doxycycline hyclate (DOX, #D9891, Sigma Aldrich) to induce NGN2 over-expression. ROCK inhibitor was removed after 24 hours. Media was changed every 24 hours with fresh complete neuron media with DOX.  On day 4 (t04), cells were passaged by Accutase and plated at 260,00 cells/cm² on PEI-coated 6-well plates in complete neuron medium with ROCK inhibitor and DOX. ROCK inhibitor was removed after 24 hours. Media was changed every 24 hours with fresh complete neuron media with DOX continued until day 7 (t07). After t07, DOX was removed, and media was changed daily until day 30 (t30).  Samples for RNA-seq were collected at day 0 (t00, iPSC), day 4 (t04, intermediate), and day 30 (t30, neuron).

value: Neuron


title: Jur_WT_PMA_ACT3

description:
Library name: sample3
Jur_WT_PMA_ACT3


title: Jur_WT_PMA_CT1

description:
Library name: sample4
Jur_WT_PMA_CT1


title: Not_Trm_PD1_pos_Tim3_pos, rep3

description:
Library name: Not_Trm_PD1_pos_Tim3_pos_3
Column name in tablecounts_normalized_lung_T_cells.csv: NotTrmPD1posTim3pos_3

value: Not_Trm_PD1pos_Tim3pos_CD8+ T cells

value: 8-12 weeks


title: Neurons replicate 1 derived from iPSC replicate 3

description: Library name: CN_3_1_150PM_CELL1


title: CHI069 Silent WT

description: Library name: CHI069

value: dorsal striatum

value: Silent

value: cholinergic interneuron

value: male

tag: date ephys

tag: dob

tag: age days

tag: date lp

tag: lp lot

tag: index lot

tag: date seq


title: stomach female high-dose exposure rep6

description: Library name: S_46

tag: batch

value: Batch 2


title: Divided_OT1_CD103pos, DC1act, rep3

description:
Library name: Divided_CD103pos_OT1_DC1act_3
Column name in tablecounts_normalized_ex_vivo_OT1.csv: DC1_CD103pos_3

tag: age


title: CHI221 Active Het

description: Library name: CHI221

tag: tissue

tag: group

value: Active

value: Het

tag: Sex

tag: electrophysiologist

tag: mouse id

tag: rt lot

tag: seq srm_sample_id


title: Dexamethasone 1

city: New York

first: Dr. Liang

last: Lei

state: NY

address: 1130 St. Nicholas Ave

department: Pathology and Cell Biology

institute: Columbia University

description: Library name: DP001

data_processing:
Libraries are sequenced using Element AVITI at Columbia Genome Center. Multiplex samples were in each lane, which yields targeted number of paired-end 75bp reads for each sample. Bases2fastq (version 1.7.0.1196148384) was used to convert BCL to fastq format, coupled with adaptor trimming. Pseudoalignment using kallisto (0.44.0) was performed. Differentially expressed genes were performed using Sleuth or DESeq2, R packages.
Assembly: Ensembl v96, Human:GRCh38.p12
Supplementary files format and content: count

source_name: Microglia

extract_protocol:
RNA was extracted with Rneasy mini kit (Qiagen)
Poly-A pull-down was used to enrich mRNAs from total RNA samples, then proceed with library construction using Illumina TruSeq chemistry. The final PCR step has been modified using the KAPA HiFi HotStart Ready Mix.

tag: cell line

value: Microglia


title: Jur_WT_PMA_ACT2

description:
Library name: sample2
Jur_WT_PMA_ACT2

value: cell line

value: Human Jurkat T cell

tag: cell type

tag: genotype

tag: treatment


title: Neurons replicate 2 derived from iPSC replicate 2

description: Library name: CN_2_2_150PM_CELL1


title: stomach female high-dose exposure rep5

description: Library name: S_45

value: stomach

tag: strain

value: Sprague-Dawley

tag: developmental stage

value: adult

value: Female-High Dose/50mg/kg


title: Divided_OT1_CD103pos, DC1act, rep2

description:
Library name: Divided_CD103pos_OT1_DC1act_2
Column name in tablecounts_normalized_ex_vivo_OT1.csv: DC1_CD103pos_2

value: Lymph node

value: KP-OVA

value: CD103pos_OT1 CD8+ T cells

value: WT


title: Neurons replicate 1 derived from iPSC replicate 2

description: Library name: CN_2_1_150PM_CELL1

value: Neuron


title: stomach female high-dose exposure rep4

description: Library name: S_44

value: Batch 1


title: Divided_OT1_CD103pos, DC1act, rep1

description:
Library name: Divided_CD103pos_OT1_DC1act_1
Column name in tablecounts_normalized_ex_vivo_OT1.csv: DC1_CD103pos_1

value: 8-12 weeks


title: Jur_WT_PMA_ACT1

description:
Library name: sample1
Jur_WT_PMA_ACT1


title: PBMC_AMG487_3

description: Library name: sample9

source_name: Human PBMCs

growth_protocol: Human PBMC cells were purchased from Shanghai Hycells Biotechnology Co., Ltd. Human PBMC cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 (Thermo Scientific) complete medium with 10% (vol/vol) fetal bovine serum (FBS, Thermo Scientific, Cat: 10270106, New York, NY, USA) and 1%(vol/vol) penicillin/streptomycin (P/S,Thermo Scientific, Cat: 15140122, New York, NY, USA).

treatment_protocol: The PMA and Ionomycin were dissolved in Dimethyl sulfoxide (DMSO) to prepare a stock solution of 320 μM and 5.2 mM, which were stored at −20 ℃. The VUF 11222 and AMG 487 were dissolved in Dimethyl sulfoxide (DMSO) to prepare a stock solution of 10 mM. Before activating the PBMCs, we pretreated them with the CXCR3 antagonist AMG 487 (10 μM) and agonist VUF 11222 (1 μM) for 6-12h, then the Human PBMC cells were stimulated with PMA/ionomycin (0.08/1.3 mM) for 12 h, followed by RNA sequencing.

value: Human PBMCs


title: Neurons replicate 2 derived from iPSC replicate 1

description: Library name: CN_1_3_150PM_CELL1


title: ChIPseq, RPB1, aux, flav, 10

tag: chip antibody

value: Colorectal carcinoma

value: Flavopiridol washout 10min


title: Divided_OT1_CD103neg, DC1act, rep4

description:
Library name: Divided_CD103neg_OT1_DC1act_4
Column name in tablecounts_normalized_ex_vivo_OT1.csv: DC1_CD103neg_4

value: CD103neg_OT1 CD8+ T cells


title: stomach female high-dose exposure rep3

description: Library name: S_43

value: Batch 2


title: PBMC_AMG487_2

description: Library name: sample8


title: Neurons replicate 1 derived from iPSC replicate 1

description: Library name: CN_1_2_150PM_CELL1


title: stomach female high-dose exposure rep2

description: Library name: S_42


title: Divided_OT1_CD103neg, DC1act, rep3

description:
Library name: Divided_CD103neg_OT1_DC1act_3
Column name in tablecounts_normalized_ex_vivo_OT1.csv: DC1_CD103neg_3


title: Intermediate cells replicate 2 derived from iPSC replicate 3

description: Library name: NPC_3_3_150PM_CELL1

value: Intermediate


title: PBMC_AMG487_1

description: Library name: sample7


title: stomach female high-dose exposure rep1

description: Library name: S_41

value: stomach

value: Batch 3


title: Divided_OT1_CD103neg, DC1act, rep2

description:
Library name: Divided_CD103neg_OT1_DC1act_2
Column name in tablecounts_normalized_ex_vivo_OT1.csv: DC1_CD103neg_2


title: stomach female low-dose exposure rep8

description: Library name: S_40

value: Sprague-Dawley

value: adult

value: Female-Low Dose/5mg/kg


title: PBMC_VUF11222_3

description: Library name: sample6


title: PBMC_VUF11222_2

description: Library name: sample5


title: Intermediate cells replicate 1 derived from iPSC replicate 3

description: Library name: NPC_3_1_150PM_CELL1


title: Divided_OT1_CD103neg, DC1act, rep1

description:
Library name: Divided_CD103neg_OT1_DC1act_1
Column name in tablecounts_normalized_ex_vivo_OT1.csv: DC1_CD103neg_1

value: Lymph node

value: KP-OVA


title: stomach female low-dose exposure rep7

description: Library name: S_39

value: WT


title: Vestibular ganglion, Tmie,female, spatial

city: beijing

first: lucinda

last: liu

state: 北京Beijing

address: 7 Science Park Road ZGC Life Science Park

institute: nibs

description: Library name: Tmie_spatial_female

data_processing:
Raw sequencing data were processed using SpaceRanger (10x Genomics, v.4.0.1)
Assembly: mouse reference genome (mm10-2020-A, refdata-gex-mm10-2020-A)
Supplementary files format and content: Tar.gz (bins matrix)
Library strategy: Spatial Transcriptomics

source_name: vestibular ganglion

extract_protocol:
Fresh vestibular ganglia tissues were dissected from 4-week-old male and female C57BL/6J wild-type (WT) and Tmie knockout (Tmie-/-) mice, embedded in OCT (Sakura) and stored at -80°C. To evaluate RNA quality, total RNA was extracted from cryosections and its integrity was assessed by measuring the RNA Integrity Number (RIN), samples with RIN > 7 were proceeded to library construction. A total of 8 sections were analyzed for the WT group (n=2, male) and 6 sections for the Tmie -/- group (n=2, female). For histological examination, 10 μm-thick cryosections were prepared using a cryostat (Leica, CM1950), mounted onto plain glass slides, fixed with formaldehyde, and subjected to hematoxylin and eosin (H&E) staining and imaging for morphological observation.
Tissue sections were hybridized with gene-specific probes (Hybridization Kit, 10x Genomics, PN-1000466/PN-1000667), followed by ligation (Ligase, PN-2000425) to stabilize probe connections. Sections were aligned to the Visium HD slide capture area (6.5X6.5 mm), and probes were transferred using the CytAssist instrument. Released probes were captured by the slide's poly-T oligos and extended to incorporate UMIs, spatial barcodes, and sequencing adapters. The barcoded cDNA was amplified, bead-purified, and ligated with dual-index adapters to construct sequencing libraries. Library concentration was quantified using a Qubit 3.0 fluorometer (Thermo Fisher, Q33216), and fragment size distribution was analyzed with an Agilent 2100 Bioanalyzer (Agilent, G2939AA). Qualified libraries were sequenced on an Illumina platform under PE150 mode to generate high-quality spatial transcriptomic data.

value: vestibular ganglion

value: Tmie ko


title: Intermediate cells replicate 2 derived from iPSC replicate 2

description: Library name: NPC_2_2_150PM_CELL1


title: PBMC_VUF11222_1

description: Library name: sample4


title: Intermediate cells replicate 1 derived from iPSC replicate 2

description: Library name: NPC_2_1_150PM_CELL1


title: stomach female low-dose exposure rep6

description: Library name: S_38

value: Batch 1


title: Vestibular ganglion, WT, male, spatial

description: Library name: WT_spatial_male

value: Wildtype


title: PBMC_CT3

description: Library name: sample3

value: Human PBMCs


title: Intermediate cells replicate 2 derived from iPSC replicate 1

description: Library name: NPC_1_3_150PM_CELL1


title: Jur_WT_CD3_CT1

description:
Library name: sample16
Jur_WT_CD3_CT1

value: cell line

value: Human Jurkat T cell


title: Not_Trm_PD1_pos_Tim3_neg, rep1

description:
Library name: Not_Trm_PD1_pos_Tim3_neg_1
Column name in tablecounts_normalized_lung_T_cells.csv: NotTrmPD1posTim3neg_1

value: Lungs

value: KP

value: Not_Trm_PD1pos_Tim3neg_CD8+ T cells

value: 8-12 weeks


title: stomach female low-dose exposure rep5

description: Library name: S_37

value: stomach

value: Sprague-Dawley


title: stomach female low-dose exposure rep4

description: Library name: S_36

value: adult

value: Female-Low Dose/5mg/kg

value: Batch 3


title: PBMC_CT2

description: Library name: sample2


title: Intermediate cells replicate 1 derived from iPSC replicate 1

description: Library name: NPC_1_1_150PM_CELL1

value: Intermediate


title: scPerturb_Aged4

first: Alex Chang-Yu

last: Chen

address: 149 13th Street, 7th floor, Room 7103

department: Krantz Family Center for Cancer Research

institute: Massachusetts General Hospital

description:
Library name: scPerturb_Aged4
10x Genomics

data_processing:
The demultiplexing, barcoded processing, gene counting and aggregation were made using the Cell Ranger software v9.0.0 (https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/what-is-cell-ranger)
Assembly: mm10
Supplementary files format and content: Gzipped files include 10x format single cell gene expression matrices, including barcodes.tsv.gz, features.tsv.gz, matrix.mtx.gz, and protospacer_calls_per_cell.csv

source_name: B16-OVA Tumor

extract_protocol:
Fifteen days after tumor inoculation, ametrine⁺ CD45.1 Cas9⁺ OT-1 CD8⁺ T cells were isolated from tumors and tumor-draining lymph nodes (tdLNs) of young and aged mice using a BD FACSAria™ Fusion sorter. Sorted cells from independent mice were pooled into two biological replicates per group, counted, and loaded onto a Chromium Controller (10x Genomics) for a target recovery of 10,000 single cells per run. Libraries were prepared with the 10x Single Cell 5′ Reagent Kits v2 (Dual Index) and Feature Barcode technology for CRISPR screening, following the manufacturer’s protocol. Sequencing was performed on an Illumina NovaSeq X or NextSeq 1000/2000 system using 100-cycle kits with paired-end reads.
Library was performed according to the manufacter’s instructions (10x Single Cell 5′ Reagent Kits v2, CG00511 RevD, 10x Genomics).

value: B16-OVA Tumor

value: Cas9 OT-1 CD8+ T cells


title: PBMC_CT1

description: Library name: sample1


title: scPerturb_Aged3

description:
Library name: scPerturb_Aged3
10x Genomics


title: scPerturb_Aged2

description:
Library name: scPerturb_Aged2
10x Genomics


title: stomach female low-dose exposure rep3

description: Library name: S_35

value: WT

value: Batch 2


title: Induced pluripotent stem cells replicate 3

description: Library name: iPSC_3_150PM_CELL1

value: iPSC


title: Induced pluripotent stem cells replicate 2

description: Library name: iPSC_2_150PM_CELL1


title: scPerturb_Aged1

description:
Library name: scPerturb_Aged1
10x Genomics


title: stomach female low-dose exposure rep2

description: Library name: S_34


title: Induced pluripotent stem cells replicate 1

description: Library name: iPSC_1_150PM_CELL1


title: stomach female low-dose exposure rep1

description: Library name: S_33


title: scPerturb_Young3

description:
Library name: scPerturb_Young3
10x Genomics


title: stomach female control rep7

description: Library name: S_31

value: Female-Vehicle Control

value: Batch 1


title: stomach female control rep8

description: Library name: S_32


title: SW480, WT, ATP treated, rep3

city: Heidelberg

first: Chong

last: Sun

state: Baden-Württemberg

address: Im Neuenheimer Feld 280

department: Immune Regulation in Cancer

country: Germany

institute: German Cancer Research Center

description:
Library name: Sample 15
Column name in all_cnt_FeatureCounts.tsv: SW480_Ctrl_3

data_processing:
Raw reads from the paired-end fastq files were aligned to GRCh38 using STAR (2.7.10a).
Raw read counts per gene were measured by FeatureCounts (2.0.1) with the default parameters.
Assembly: GRCh38
Supplementary files format and content: tab-delimited text file including raw read counts for each sample

growth_protocol: KBM7 cells were cultured in IMDM, while BxPC3 and SW480 cells were maintained in RPMI 1640 medium. All media were supplemented with 10% fetal bovine serum, 100 U/mL penicillin-streptomycin, and GlutaMAX. The cells were incubated in a humidified atmosphere at 37 °C with 5% CO₂.

extract_protocol:
RNeasy Mini Kit (QIAGEN) was used for RNA isolation. 1μg total RNA was used for the construction of sequencing libraries.
RNA libraries were constructed using the Hieff NGS Ultima Dual-Mode mRNA Library Prep Kit for Illumina (Yeasen, model: 13533ES96), following the manufacturer's protocol.

treatment_protocol: All the cells were treated with 200 μM ATP for 8 hours.

value: Colorectal cancer cells

value: 200 μM ATP


title: scPerturb_Young2

description:
Library name: scPerturb_Young2
10x Genomics


title: SW480, WT, ATP treated, rep2

description:
Library name: Sample 14
Column name in all_cnt_FeatureCounts.tsv: SW480_Ctrl_2


title: MDA-MB-231_10kpa

city: Québec

first: Martial

last: Millet

state: Québec

address: 2250, Boulevard Henri-Bourassa

institute: CHU de Québec - Université Laval

description:
Library name: Sample 2
Colname in processed files : MDA_10kPa

data_processing:
Raw fast5 files were basecalled using Guppy v.4.2.2 with the guppy_basecaller command and the dna_r9.4.1_450bps_fast.cfg configuration file and default settings. Barcodes were detected and reads were separated with guppy_barcoder using the default settings. The resulting FASTQ reads were aligned to the GRCh38 human reference with Minimap2 v2.17-r941 (Li, 2018, https://doi.org/10.1093/bioinformatics/bty191) using the following parameters: -aLx splice –cs = long.
Raw sequencing reads (FASTQ format) were processed using fastp (v0.23.4) for quality control and adapter trimming with default parameters, including automatic detection of paired-end adapters, trimming of bases with Phred quality scores <20, removal of reads containing >30% low-quality bases, and exclusion of reads shorter than 30 bp. High-quality reads were aligned to the human reference genome (GRCh38) using STAR (v2.7.3a) with default settings, and resulting alignments were retained as coordinate-sorted BAM files.
Alternative splicing events were quantified using PSI-Sigma (v2.1), a junction-based approach for estimating Percent Spliced In (PSI) values from RNA-seq data. PSI-Sigma was executed via the dummyai.pl script with a minimum read support threshold of five reads per event (--nread 5). Splicing analyses were performed using the human reference genome (GRCh38) .
Assembly: GRCh38
Supplementary files format and content: PSI_MDA-MB231_MCF10a_Stiffness.csv : Alternative splicing events detected using PSI-Sigma.

growth_protocol: Highly metastatic MDA-MB-231 breast adenocarcinoma cells (HTB-26, ATCC) were maintained in DMEM (Wisent Bioproducts #319-015-CL) supplemented with 10% fetal bovine serum (FBS) (Thermo Scientific # A5256701) and 1% penicillin-streptomycin (Wisent Bioproducts #450-201-EL). MCF10a mammary epithelial cells (ATCC) were maintained in DMEM/F12 (Wisent Bioproducts #319-085-CL) supplemented with 5% horse serum, 20 ng/ml EGF (Invitrogen, Carlsbad, CA), 10 mg/ml insulin, 0.5 mg/ml hydrocortisone, 100 ng/ml cholera toxin (Sigma-Aldrich) and 1% penicillin–streptomycin (Wisent Bioproducts #450-201-EL). Both cell lines were cultured at 37°C and 5% CO2.

extract_protocol:
After 72h and 48 h culture on polyacrylamide gels respectively, MDA-MB-231or MCF10a were rinsed with PBS, and collected after being treated with 0.05% and 0.25% trypsin respectively. Cell lysates were homogenized with QIAshredder columns (QIAGEN #79654) and total RNA was extracted with RNeasy mini columns (QIAGEN #74104). RNA was DNase treated using TURBO DNase (ThermoFisher Scientific #AM2238). RNA quality was verified on a 2100 Bioanalyzer (Agilent Technologies) and polyadenylated RNA was enriched using NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB #E7490L).
Libraries were generated using 100 ng of poly(A)+ RNA and Oxford Nanopore Technologies’ Direct cDNA Sequencing Kit (ONT SQK-DCS109), with each sample barcoded with the Native Barcoding Expansion (ONT EXP-NBD104), according to the manufacturer’s specifications.
Barcoded samples were pooled, loaded on a MinION sequencer using four R9 Flow Cells (ONT FLO-MIN106D), and sequenced using the MinKNOW software v19.

value: Triple-negative breast cancer cell line


title: MCF10a_10kpa

description:
Library name: Sample 4
Colname in processed files : MCF10a_10kPa

value: Non-tumorigenic mammary epithelial cell line


title: stomach female control rep6

description: Library name: S_30

value: stomach


title: scPerturb_Young1

description:
Library name: scPerturb_Young1
10x Genomics


title: SW480, WT, ATP treated, rep1

description:
Library name: Sample 13
Column name in all_cnt_FeatureCounts.tsv: SW480_Ctrl_1


title: MDA-MB-231_1kpa

description:
Library name: Sample 1
Colname in processed files : MDA_1kPa


title: stomach female control rep5

description: Library name: S_29

value: Sprague-Dawley


title: IFNgKO4

city: Montreal

first: Ana Maria

last: Hincapie

address: 1160 Pine avenue West

institute: McGill University

description: Library name: C008

data_processing:
SALMON
Assembly: MM_grch38_ens94
Supplementary files format and content: .xlsx

source_name: Isolated B lymphocytes

extract_protocol:
Qiagen Rneasy Plus Universal mini kit
NEBNext Ultra II RNA Library Prep Kit for Illumina

value: Isolated B lymphocytes

value: Ptpn2fl/fl mb1 Cre+

value: IFN-gamma 6 hour treatment


title: BxPC-3, P2RY2 KO, ATP treated, rep3

description:
Library name: Sample 12
Column name in all_cnt_FeatureCounts.tsv: BxPC3_KO_3

value: Pancreatic ductal adenocarcinoma cells

value: P2RY2 knockout


title: IFNgKO3

description: Library name: C007


title: stomach female control rep4

description: Library name: S_28

value: adult


title: BxPC-3, P2RY2 KO, ATP treated, rep2

description:
Library name: Sample 11
Column name in all_cnt_FeatureCounts.tsv: BxPC3_KO_2


title: stomach female control rep3

description: Library name: S_27

value: WT


title: SEM_USP22-ChIP_rep2

city: Hannover

first: Florian

last: Perner

address: Carl-Neuberg-Str. 1

institute: Hannover Medical School

description: Library name: SEM_USP22-ChIP_rep2

data_processing:
Data processing and analysis was perfomed in a Galaxy environment (version: 24.2.3) hosted at Hannover Medical School, Department of Hematology, Hemostasis, Oncology and Stem Cell Transplantation
FASTQ files were trimmed using "fastp" (0.23.4) (default mode)
Trimmed paired-end reads were aligned to the human reference genome hg19 using Bowtie2 (2.5.3).
Read counts were obtained using bedtools multicov (2.30.0), and coverage tracks were generated using bamCoverage (3.5.4) for visualization in IGV (2.17.4).
Duplicates were removed using macs2 filterdup (2.2.9.1) and peaks were called with macs2 callpeak (2.2.9.1)
Assembly: hg38
Supplementary files format and content: .bigwig file encoding coverage data for visualization in genome browser for all samples
Supplementary files format and content: .bed files containing the peak calling results for each ChIP sample

source_name: hematopoietic

growth_protocol: The human leukemia cell lines MV4;11 and SEM were grown in RPMI+10%FBS+5%Pen/Strep (Gibco) at 5% CO2 and 37°C.

extract_protocol:
Cells were double cross-linked with 50 µL/mL of 40 mM disuccinimidyl glutarate (DSG) for 30 minutes and 2% formaldehyde solution for 10 minutes. The reaction was quenched by adding 200 µL of 1.25 M glycine and 100 µL of 1 M Tris-HCl per mL of suspension. Cells were pelleted by centrifugation (1400 rpm, 5 min). Cells were lysed in 1 mL of Cytoplasmic Lysis Buffer (20 mM Tris-HCl pH 7.5, 300 mM NaCl, 2 mM EDTA, 0.5% NP-40, 1% Triton X-100; freshly supplemented with 1 mM PMSF and cOmplete™ Protease Inhibitor Cocktail). Nuclei were extracted by applying 10–15 strokes with a pre-chilled Dounce homogenizer. The nuclear pellet was resuspended in Sonication Buffer (10 mM Tris-HCl pH 8.1, 100 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.5% N-lauroylsarcosine, 0.1% SDS; freshly supplemented with 1 mM PMSF and cOmplete™ Protease Inhibitor Cocktail). Chromatin was sonicated to an average fragment size of 200–600 bp using a Covaris ME220 Focused-ultrasonicator. For immunoprecipitation, 5 µg of antibody per IP were used. Beads were then resuspended in Wash Buffer (50 mM HEPES pH 7.6, 500 mM LiCl, 1 mM EDTA, 1% NP-40, 0.7% sodium deoxycholate; freshly supplemented with 1 mM PMSF) and washed six times with 800 µL Wash Buffer by gentle inversion for 2 minutes per wash, followed by a final wash with 1 mL of 1x TE buffer. Chromatin was eluted in 150 µL Elution Buffer and decrosslinked at 65°C overnight.
Library preparation for Illumina sequencing was performed using the NEBNext Ultra II DNA Library Prep Kit, following the manufacturer's instructions. Libraries were purified with 0.9× AmpureXP beads

treatment_protocol: ChIPseq was performed under steady-state conditions. No drug treatment was applied.

value: hematopoietic

value: SEM

value: ALL

value: anti-USP22, Novus Biologicals, cat:NBP1-49644, lot:A-9


title: SEM_USP22-ChIP_rep1

department: Hematology, Hemostasis, Oncology and Stem Cell Transplantation

description: Library name: SEM_USP22-ChIP_rep1


title: IFNgKO2

description: Library name: C006


title: BxPC-3, P2RY2 KO, ATP treated, rep1

description:
Library name: Sample 10
Column name in all_cnt_FeatureCounts.tsv: BxPC3_KO_1


title: SEM_MEIS1-ChIP_rep2

description: Library name: SEM_MEIS1-ChIP_rep2


title: stomach female control rep2

description: Library name: S_26

value: Female-Vehicle Control

value: Batch 2


title: IFNgKO1

description: Library name: C005


title: BxPC-3, WT, ATP treated, rep3

description:
Library name: Sample 9
Column name in all_cnt_FeatureCounts.tsv: BxPC3_Ctrl_3

value: 200 μM ATP


title: SEM_MEIS1-ChIP_rep1

description: Library name: SEM_MEIS1-ChIP_rep1


title: BxPC-3, WT, ATP treated, rep2

description:
Library name: Sample 8
Column name in all_cnt_FeatureCounts.tsv: BxPC3_Ctrl_2


title: IFNgWT4

description: Library name: C004

value: Ptpn2wt/wt mb1 Cre+


title: stomach female control rep1

description: Library name: S_25

value: stomach

value: Batch 1


title: IFNgWT3

description: Library name: C003

value: Isolated B lymphocytes


title: stomach male high-dose exposure rep8

description: Library name: S_24

value: Sprague-Dawley

value: Male-High Dose/50mg/kg

value: Batch 3


title: SEM_input

description: Library name: SEM_input


title: BxPC-3, WT, ATP treated, rep1

description:
Library name: Sample 7
Column name in all_cnt_FeatureCounts.tsv: BxPC3_Ctrl_1

value: Pancreatic ductal adenocarcinoma cells


title: MV411_USP22-ChIP_rep2

description: Library name: MV411_USP22-ChIP_rep2

value: AML

value: anti-MEIS1, Abcam, cat:AB19867, lot:1093686-2


title: stomach male high-dose exposure rep7

description: Library name: S_23

value: adult

value: WT


title: IFNgWT2

description: Library name: C002

value: IFN-gamma 6 hour treatment


title: KBM7, P2RY2 KO, ATP treated, rep3

description:
Library name: Sample 6
Column name in all_cnt_FeatureCounts.tsv: KBM7_KO_3

value: Chronic myelogenous leukemia cells

value: P2RY2 knockout


title: KBM7, P2RY2 KO, ATP treated, rep2

description:
Library name: Sample 5
Column name in all_cnt_FeatureCounts.tsv: KBM7_KO_2


title: MV411_MEIS1-ChIP_rep2

description: Library name: MV411_MEIS1-ChIP_rep2

value: hematopoietic


title: stomach male high-dose exposure rep6

description: Library name: S_22

value: Batch 2


title: MV411_USP22-ChIP_rep1

description: Library name: MV411_USP22-ChIP_rep1


title: IFNgWT1

description: Library name: C001

value: Ptpn2wt/wt mb1 Cre+


title: stomach male high-dose exposure rep5

description: Library name: S_21

value: stomach


title: KBM7, P2RY2 KO, ATP treated, rep1

description:
Library name: Sample 4
Column name in all_cnt_FeatureCounts.tsv: KBM7_KO_1

value: 200 μM ATP


title: MV411_MEIS1-ChIP_rep1

description: Library name: MV411_MEIS1-ChIP_rep1


title: KBM7, WT, ATP treated, rep3

description:
Library name: Sample 3
Column name in all_cnt_FeatureCounts.tsv: KBM7_Ctrl_3


title: MV411_input

description: Library name: MV411_input


title: stomach male high-dose exposure rep3

description: Library name: S_19


title: aIgMKO2

description: Library name: B006

value: Isolated B lymphocytes

value: Ptpn2fl/fl mb1 Cre+

value: anti-IgM 6 hour treatment


title: aIgMKO1

description: Library name: B005


title: KBM7, WT, ATP treated, rep1

description:
Library name: Sample 1
Column name in all_cnt_FeatureCounts.tsv: KBM7_Ctrl_1


title: stomach male high-dose exposure rep2

description: Library name: S_18

value: Sprague-Dawley

value: Male-High Dose/50mg/kg


title: Pituitary cells

city: Shanghai

first: Cui

last: Zhang

address: 197, Ruijin 2nd Road

institute: Ruijin Hospital

description:
Library name: Sample1
10x Genomics

data_processing:
Raw sequencing data were processed using Cell Ranger (v5.0.1) for alignment to the GRCh38-2020-A reference genome and gene expression quantification. Quality control and filtering included removal of genes expressed in less than 1% of cells, retention of cells with 300–4,500 detected genes (the upper threshold representing 90% of the maximum gene count per cell), doublet detection and removal using the doubletdetection Python package, and exclusion of cells with high mitochondrial gene content (top 15%).
Assembly: GRCh38-2020-A
Supplementary files format and content: Tab-separated values files and matrix files

molecule: nuclear RNA

source_name: Pituitary

extract_protocol:
Nuclei were isolated from a CCS pituitary tumor sample previously stored in liquid nitrogen using a mortar-based homogenization method, followed by purification via iodixanol (Sigma, D1556-250ML) density gradient centrifugation. Quality control included nuclei counting with AOPI staining on a Countstar S2 instrument and assessment of nuclei integrity by trypan blue staining under a microscope.
Library construction was performed using the Chromium Next GEM Single Cell 3' Kit v3.1 (10X Genomics) on the Chromium Controller platform. Approximately 25,410 nuclei (30 µL at 847 nuclei/µL) were loaded, targeting 10,000 nuclei capture. Gel Beads-in-emulsion were generated with near-saturation loading of barcoded beads, enabling hybridization of polyadenylated RNA to the beads upon lysis. Reverse transcription was carried out to synthesize cDNA tagged with cell barcodes and unique molecular identifiers (UMIs), followed by second-strand synthesis, adapter ligation, and universal amplification according to the manufacturer's protocol. Libraries were quantified using an Agilent 2100 Bioanalyzer with a High Sensitivity DNA Chip and a Qubit High Sensitivity DNA Assay kit (Thermo). Sequencing was performed on an Illumina NovaSeq X Plus platform in PE150 mode, targeting 20,000 reads per nucleus and a total output of 100 Gb.

value: Pituitary

value: tumor cell

value: female


title: KBM7, WT, ATP treated, rep2

description:
Library name: Sample 2
Column name in all_cnt_FeatureCounts.tsv: KBM7_Ctrl_2

value: WT


title: CAL27,FOXK1 knockout,rep2

city: Stanford

first: Namyoung

last: Jung

state: CA

address: 269 Campus Dr. CCSR Room 2150

department: Medicine

institute: Stanford University

description: Library name: CAL27_FOXK1_KO_rep2

data_processing:
Single-end reads were aligned using STAR aligner (2.5.4b) using GENCODE v19 gene annotation in hg19 with default settings.
To obtain raw expected read counts, RSEM (1.3.0) was used, and these raw counts were subjected to differential expression analysis with DESeq2 (1.26.0).
Assembly: hg19
Supplementary files format and content: tab-delimited text file includes expected counts from RSEM for each sample

growth_protocol: CAL27 human squamous cell carcinoma cells were obtained from ATCC (CRL-2095) and cultured in DMEM F:12 (ThermoFisher) supplemented with 10% FBS. SCC-13 human squamous cell carcinoma cells were obtained from J.G.Rheinwald, Dana-Farber/Harvard Cancer Center as a generous gift and cultured in Keratinocyte-SFM (ThermoFisher) supplemented with 25 ug/ml BPE, 0.2ng/ml EGF (GIBCO, Thermofisher) and 0.3mM CaCl2 (Sigma-Aldrich). Primary human keratinocytes were isolated and cultured from fresh, surgically discarded neonatal foreskin by protocols approved by the Stanford Human Subjects Institutional Review Board and in accordance with the NIH genomic data sharing policy. Keratinocytes were cultured in 1:1 Keratinocyte-SFM (ThermoFisher) and Medium 154 (ThermoFisher) supplemented with HKGS (ThermoFisher). Keratinocytes were induced to differentiate by the addition of 1.2mM calcium for 3 days at full confluence.

extract_protocol:
Cells were harvested and pellets were flash frozen on dry ice. RNA was extracted using the RNeasy Mini Kit (QIAGEN).
RNA-seq libraries were generated on two biological replicates for each group using the Lexogen Quant-seq 3’ mRNA -seq Library Prep Kit FWD (Lexogen) according to the manufacturer’s protocol. The libraries were sequenced as single-end 1X101bp reads on an Illumina HiSeq2000 or 1X150bp reads on an Illumina NovaSeq 6000 instrument.

treatment_protocol: Dharmacon siStable siRNA was designed to target PAN-p63 with the sequences as follows: sense (5’-CGACAGUCUUGUACAAUUUUU-3’) and antisense (5’-AAAUUGUACAAGACUGUCGUU-3’) or FOXK1 with si1 sense (5’-GAACGGAAAGCAUGCGGUU-3’), antisense (5’-AACCGCAUGCUUUCCGUUC-3’) and si2 sense (5'-GCAGUGUACCUUCCGGUUU-3’) and antisense (5’-AAACCGGAAGGUACACUGC-3’). 1-1.5nmol of siRNA was electroporated into 1 million keratinocytes and 2 million SCC cells using the human keratinocyte Nucleofector kit (Lonza) according to the manufacturer’s protocol. After 24 hours, fresh media was added to the cells and cells were subsequentially seeded for differentiation after 48 h transfection or maintained at subconfluence. The negative control was the ON-TARGET Smart pool negative control (Horizon, D-001810-10-20).

value: Squamous cell carcinoma

value: FOXK1 knockout


title: MCF10a_1kpa

description:
Library name: Sample 3
Colname in processed files : MCF10a_1kPa

value: Non-tumorigenic mammary epithelial cell line


title: stomach male high-dose exposure rep1

description: Library name: S_17

value: adult

value: Batch 3


title: scPerturb_Young4

description:
Library name: scPerturb_Young4
10x Genomics

value: B16-OVA Tumor

value: Cas9 OT-1 CD8+ T cells


title: SW480, P2RY2 KO, ATP treated, rep1

description:
Library name: Sample 16
Column name in all_cnt_FeatureCounts.tsv: SW480_KO_1

value: Colorectal cancer cells

value: P2RY2 knockout

value: 200 μM ATP


title: aIgMWT4

description: Library name: B004

value: Isolated B lymphocytes

value: Ptpn2wt/wt mb1 Cre+

value: anti-IgM 6 hour treatment


title: IFN15_12months_cd34pos_GoT-ChA

first: Anna

middle: S.

last: Nam

state: New York

address: Weiss Research Building, Floor 11

department: Pathology and Laboratory Medicine

institute: Weill Cornell Medicine

description:
Library name: IFN15_12months_cd34pos_GoT-ChA
GoT-ChA

data_processing:
10x data was processed using Cell Ranger 1 and v6.1.2 using default parameters. RNA reads were aligned to the human referece genome. Amplicon reads was processed following steps: 1) Identification of reads with proper priming, 2) Identification of cell barcodes (CB) that match with the whitelisted CB, 3) Replacement of CB that are not perfectly matched with the whitelist CB, 4) Deduplication of reads, 5) Analyze reads with CB that are also observed in the 10x RNA-seq dataATAC-seq 6) Collapse networks of closely related UMIs within CBs to correct for sequencing error, 7) Matching of CB/UMI pairs found in 10x scRNA-seq data, discarding those matching non-CALR genes and those matching no gene below a supporting read threshold.
CUT&RUN data were down-sampled to have same number of reads across all conditions within the sample replicate to account for difference in sequencing depth. Among the down-sampled reads, low-quality reads were filtered out using Trimmomatic, resulting reads were aligned with hg38 genome with Bowtie2, and PCR duplicates were removed with SAMtools with a previously described workflow. Aligned reads were then used for peak calling with MACS3 with a q-value threshold of 0.01 and reads from IgG antibody as the control. SPI1 over-enrichment in the called peaks was confirmed with Simple Enrichment Analysis (SEA, v5.5.5). Overlapping peaks between replicates of the same condition were kept for further processing. The peaks were processed with multiBamSummary function from deepTools to obtain counts matrix per sample for downstream analysis.
Genotyping data processing was performed according to the previously published instructions and available Gotcha R package. Briefly, initial FASTQ files were filtered based on base quality scores, and the Fastqsplit function was used to split FASTQ files for subsequent parallel computing. Mutations were determined via the BatchMutationCalling function, which identifies reads that are correctly primed, calculates the counts of reads per cell for each specified genotype (i.e., WT, JAK2-V617F).
Assembly: GRCh38
Supplementary files format and content: Processed 10X data is matrix table of gene/peak counts per cell for each sample
Supplementary files format and content: Output matrices processed from genotyping data have two columns: barcodes (BC) and Genotype

source_name: Bone marrow

extract_protocol:
GoT-IM Extraction Protocol: Cryopreserved bone marrow mononuclear cells approved by the Institutional Review Board of Weill Cornell Medicine were thawed and stained using standard procedures with the surface antibody CD34-PE (clone AC136, dilution 1:50, Miltenyi Biotec) and DAPI (Sigma-Aldrich), according to manufacturer’s protocol. To eliminate experimental batch effects, cells were labelled simultaneously with hashing antibodies with time-point-identifying barcodes using Hashtag Antibodies 1, 2 or 3 (TotalSeq-A, clone LNH-94, Biolegend) for GoT-IM. To link cell identities to expression of cell surface proteins, cells were also incubated with CITE-seq antibodies according to manufacturer protocol (TotalSeq-A, Biolegend). Cells were subsequently sorted for DAPI−, CD34+ cells using BD Influx at the Weill Cornell Medicine flow cytometry core. Genotyping of Transcriptomes (GoT) was performed by adapting the 10x Genomics platform. FACS-sorted CD34+ cells for each time-point from the same individual were pooled, targeting a total of 10,000-15,000 cells.  GoT-ATAC Extraction Protocol: Cryopreserved bone marrow mononuclear cells were thawed and stained using standard procedures with the surface antibody CD34-PE (clone AC136, dilution 1:50, MACS) and DAPI (Sigma-Aldrich), according to manufacturer’s protocol. Cells were subsequently sorted for DAPI−, CD34+ cells using BD Influx at the Weill Cornell Medicine flow cytometry core. Nuclei were isolated from DAPI-, CD34+ cells according to 10x Genomics Demonstrated Low Cell Input Nuclei Isolation protocol. Lysis buffer was prepared following manufacturer’s recommendations and then split into aliquots for each serial sample. Either TotalSeq-A Anti-Nuclei Pore Complex Proteins Hashtag 9 or 10 Antibody (1uL at 1:5 dilution; BioLegend) was added to each aliquot of lysis buffer. Low-input nuclei isolation was otherwise performed following manufacturer’s recommendations. Subsequently, nuclei from each time-point were counted and pooled together at approximately equal proportions. For IFN03, additional nuclei from the IFNa-treated sample were available to be run on a separate lane.  RFX3-OE Extraction Protocol: Cells from frozen umbilical cord blood mononuclear cells were thawed rapidly at 37°C, red blood cell lysis was performed using ACK Lysis Buffer, then centrifuged, and cell pellet was resuspended in cold MACS buffer. Cells were centrifuged at 300g for 5min at 4°C, and resuspended in cold MACs buffer at a concentration of 1x10^7 cells/mL. CD34+ cells were then isolated with the EasySep™ Human CD34 Positive Selection Kit II (StemCell Technologies #17856) according to manufacturer’s protocol. After isolation, cells were counted and plated in 96-well round-bottom plates (Falcon) in StemSpan™ SFEM II media (StemCell Technologies) with StemSpan™ CD34+ Expansion Supplement (StemCell Technologies) and 1% Pen/Strep. After 24 hours in culture, cells were spun down and resuspended in minimal media consisting of StemSpan StemSpan™ SFEM II media with StemSpan™ CD34+ Expansion Supplement, 1% Pen/Strep, 10μM prostaglandin E2 (StemCell Technologies #72192) and 100ng/μl poloxamer 407 (Millipore Sigma P2164030). Cells were split into 2 technical replicates each for untransduced condition, mCherry-control vector transduction, and RFX3-OE vector transduction. Lentivirus or PBS control was added at an MOI of 100, spinoculated at 300g, 32°C for 1 hour, and cells were incubated in same lentivirus-containing media for 24 hours at 37°C before resuspension in fresh media consisting of StemSpan™ SFEM II media with StemSpan™ CD34+ Expansion Supplement and 1% Pen/Strep. After 48 hours of incubation, cells were stained with CD34-APC antibody (1:50). Cells were then FACS isolated for the CD34+ population in the untransduced condition and the CD34+ mCherry+ populations in the RFX3-OE and mCherrycontrol conditions. Three independent transduction experiments were completed with new units of umbilical cord blood cells. Untransduced, mCherry-Control, and RFX3-OE CD34+ cells were stained with Cell Hashing antibodies (HTO TotalSeq-A, BioLegend) for 30 min at 4°C. Cells were then washed with FACs Buffer three times, pooled, and counted for loading. scRNA-sequencing was then performed using the 10x 3’ v3.1 platform according to manufacturer’s protocol. HTO demultiplexing and downstream analyses such as differential gene expression and gene set enrichment were performed as in GoT-IM for CD34+ bone marrow samples. scRNA-seq experiment was completed on one replicate of three independent transduction experiments.  CUT&RUN Extraction Protocol: UT7 cell lines expressing MPL (thrombopoietin receptor) and either the mutant CALR (type 1, L367Tfs*46) or wildtype CALR transgene (93) were seeded in 10cm plates in DMEM supplemented with 10% FBS (Thomas Scientific) and 5 ng/ml GM-CSF (Miltenyi-Biotec). Cells were treated with 0.1μg/ml Recombinant Human IFN-alpha2a (RC217-14) or PBS-1X control for 24hrs. After treatment, cells were washed 1x with PBS and collected for use with the CUTANA™ CUT&RUN assay (EpiCypher) according to the manufacturer’s protocol. 0.5μg of anti-human rabbit monoclonal PU.1 antibody (ab76543) and IgG antibody (ab172730) were used in the assay.
GoT-IM Library Construction Protocol: The standard 10x Genomics Chromium 3′ (v.3.1 chemistry) was implemented according to the manufacturer’s recommendations up to the cDNA amplification step (10X Genomics, Pleasanton, CA). After cDNA amplification and SPRI cleanup, 10x scRNA-seq and ADT/HTO libraries were generated as recommended. A portion of the cDNA was used for somatic genotyping. Briefly, to capture the somatic genotypes of cells, cDNA was amplified with a locus-specific amplification (10 PCR cycles), using the generic forward SI-PCR primer and a following locus-specific reverse primer for the CALR mutations. The amplified locus-specific cDNAs are then cleaned using SPRI purification to remove unincorporated primers. Finally, the targeted amplicon libraries are generated through a PCR performed with a P5 generic forward PCR primer together with an RPI-x primer. The targeted amplicon libraries were spiked into the remainder of the gene expression and immunophenotyping libraries to be sequenced together on a NovaSeq (Illumina, San Diego, CA). The cycle settings were as follows: 28 cycles for Read 1, 90 cycles for Read 2, 8 cycles for i7 sample index and 8 cycles for i5 sample index.  GoT-ATAC Library Construction Protocol: Single-nucleus gene expression (GEX) and chromatin accessibility libraries were constructed from the pooled nuclei according to the Chromium Next GEM Single Cell Multiome User Guide (10X Genomics). Genotyping libraries targeting the CALR mutant transcripts were constructed from the remaining amplified cDNA, similar to the original GoT method. The re-amplified sample was purified and concentrated via 0.7X SPRI cleanup, eluting it into 10uL Buffer EB. To pre-enrich the CALR mutation locus, a gene-specific PCR was performed with 3uL of cleaned re-amplified cDNA and Partial Read 1 and gene-specific primers. CALR locus-specific amplification was then performed with 3uL of cleaned gene-specific amplified cDNA and SI-PCR and loci-specific Primers. A 0.7X SPRI cleanup was performed, and cDNA was eluted into 11uL Buffer EB. Finally, to construct the targeted amplicon library, loci-amplified cDNA was mixed with P5 Generic and RPIx indexing primers and amplified. The constructed library was cleaned via 0.8X SPRI cleanup and eluted into 12uL Buffer EB. At the cDNA amplification stage of the Chromium Next GEM Single Cell Multiome protocol, supernatant from the 0.6X size selection was retained and was used to generate the hashing libraries as per HTO protocol25 with the following modification. For the hashing library construction step, the PCR reaction was prepared with 0.65uL of 10uM SI-PCR primer, 0.65uL of 10uM TruSeq DNA D7xx_s primer, 11.25uL cleaned supernatant and 12.5 uL Kapa HiFI HotStart Ready Mix (Roche, Basel, Switzerland). Hashing, gene expression, and genotyping libraries were pooled and sequenced together on a NovaSeq (Illumina).  Genotyping for JAK2-mutated samples (IFN14, IFN15): Nuclei were processed according to the 10x Genomics Chromium Next GEM Single-Cell ATAC v2 Kit guidelines, with several modifications specific to the GoT-ChA protocol. 1 µl of 22.5 µM GoT–ChA primer mix was added to the barcoding reaction mixture, containing the locus-specific primers GoT–ChA_R1N and GoT–ChA_Rev (primer sequences in Supplementary Table 32). During the post-GEM incubation clean-up, 45.5 µl of elution solution I was used to elute the SPRIselect bead reaction, and 5 µl was used for GoT-ChA library generation. GoT-ChA libraries were generated according to the published protocol.

value: Bone marrow

tag: disease state

value: Essential thrombocythemia

value: IFNa


title: CAL27,FOXK1 knockout,rep1

description: Library name: CAL27_FOXK1_KO_rep1


title: aIgMWT3

description: Library name: B003


title: CAL27,Control,rep2

description: Library name: CAL27_CTRL_rep2

value: WT


title: IFN15_12months_cd34pos_sn10x

description:
Library name: IFN15_12months_cd34pos_sn10x
10x genomics

library_source: genomic single cell


title: stomach male low-dose exposure rep8

description: Library name: S_16

value: stomach

value: Sprague-Dawley

value: Male-Low Dose/5mg/kg

tag: batch

value: Batch 2


title: aIgMWT2

description: Library name: B002


title: stomach male low-dose exposure rep7

description: Library name: S_15

value: adult


title: IFN15_Baseline_cd34pos_GoT-ChA

description:
Library name: IFN15_Baseline_cd34pos_GoT-ChA
GoT-ChA

value: Control


title: stomach male low-dose exposure rep6

description: Library name: S_14

value: Batch 1


title: CAL27,Control,rep1

description: Library name: CAL27_CTRL_rep1

value: Squamous cell carcinoma


title: NSKO4

description: Library name: A008

value: Ptpn2fl/fl mb1 Cre+

value: No treatment


title: stomach male low-dose exposure rep5

description: Library name: S_13