title: D6_Teff_Stim_high_IL2RA_screen

data_processing:
All pooled screens were analyzed with MAGeCK (v0.5.9.5). MAGeCK count was performed on all donors using --norm-method none followed by MAGeCK test --sort-criteria pos to identify genes that resulted in a statistically significant change in IL2RA expression. Results are calculated as the IL2RA low bin/IL2RA high bin.
Assembly: NA
Supplementary files format and content: Gene level and sgrna level MAGeCK results for IL2RA screens with combined donors calculated as the IL2RA low bin/IL2RA high bin.

source_name: Blood

growth_protocol: CD4+ effector T cells were isolated from fresh Peripheral Blood Leukopaks (STEMCELL Technologies, #70500) from healthy human donors, after institutional review board–approved informed written consent (STEMCELL Technologies). The contents of the Leukopaks were washed twice with a 1X volume of EasySep buffer (DPBS, 2% fetal Bovine Serum (FBS), 1mM pH 8.0 EDTA) using centrifugation. The washed cells were resuspended at 200E6 cells/mL in EasySep buffer and isolated with the EasySep™ Human CD4+CD127lowCD25+ Regulatory T Cell Isolation Kit (STEMCELL Technologies, #18063), according to the manufacturer’s protocol. Teffs were seeded at 1x106 cells/mL in RPMI-1640 supplemented with 10% FCS, 2 mM L-Glutamine (Fisher Scientific #25030081), 10 mM HEPES (Sigma, #H0887-100ML), 1X MEM Non-essential Amino Acids (Fisher, #11140050), 1 mM Sodium Pyruvate (Fisher Scientific #11360070), 100 U/mL Penicillin-Streptomycin (Sigma, #P4333-100ML), and 50 U/mL IL-2 (Amerisource Bergen, #10101641). Cells were then stimulated with ImmunoCult™ Human CD3/CD28/CD2 T Cell Activator (STEMCELL Technologies, #10990) at 6.25 uL/mL for Teff. Following activation and electroporation, cells were split 1:2 every 48 hours to maintain an approximate density of 1x106 cells/mL and supplemented with IL-2.

extract_protocol:
Cell sorting was performed 10 days following isolation for the resting screens. For the stimulated Teff screen, cells were restimulated with ImmunoCult™ Human CD3/CD28/CD2 T Cell Activator (STEMCELL Technologies, #10990) 9 days following initial isolation and sorting was performed 72 hours after restimulation, at the time of peak IL2RA expression. Prior to sorting, cells were counted, washed once with EasySep buffer, and stained with Alexa Fluor® 647 anti-human IL2RA (CD25) Antibody (Biolegend, #302618). Cells were then washed and resuspended in EasySep buffer. During sorting, cells were gated on the GFP+ population (lentiviral guide library marker) and the top and bottom 20% of IL2RA expressing cells were sorted into 15 mL conical tubes coated with FCS. Isolated cells were pelleted, counted and lysed. gDNA extraction was performed using phenol-chloroform extractions.
sgRNA was amplified and barcoded from the genomic DNA according to the protocol by Joung et al. (Joung et al. 2017). Up to 2.5 µg of genomic DNA were added to each 50 µL reaction, which included 25 µL of NEBNext Ultra II Q5 master mix (NEB, Cat #M0544L), 1.25 µL of the 10 µM forward primer and 1.25 µL of the 10 µM reverse primer, and H2O to 50 uL. The following PCR cycling conditions were used: 98°C for 3 minutes, followed by 23 cycles at 98°C for 10 seconds, 63°C for 10 seconds, and 72°C for 25 seconds, and ending with 2 minutes at 72°C. Samples were purified using 1X SPRI. The concentration of each sample was then measured using the Qubit dsDNA high sensitivity assay kit (Thermo Fisher Scientific, Cat #Q32854).
Libraries were sequenced on an Illumina HiSeq 4000 at the UCSF CAT using a custom sequencing primer.

treatment_protocol: Pooled screens were performed following the protocol described in Freimer et al 2022. In brief, 24 hours after stimulating and plating the T cells, the trans-regulator lentiviral library25 was added to each culture. The cells were counted prior to transduction, and virus was added at a multiplicity of infection (MOI) of 0.8, using gentle mixing to disperse the viral media without disrupting cell bundling. The cells were then incubated at 37°C for an additional 24 hours, pelleted by centrifugation, and viral media was replaced with fresh media supplemented with IL-2. 24 hours after washing, the cells were pelleted by centrifugation at 150 g for 10 minutes, resuspended at 1.5 x106 cells per 17.8 µL supplemented P3 Primary Cell Nucleofector Buffer (Lonza, component of #V4SP-3960) and combined with 7.2 µL RNP/1.5 x106 cells in a sterile 10 mL reservoir. After mixing the cells and RNPs, 25 µL of the mixture was distributed to the wells of a 96-well Nucleocuvette Plate (Lonza, component of #V4SP-3960). Cells were nucleofected using code EO-115 for Tregs and EH-115 for Teffs on the Lonza 4D-Nucleofector System with the 96-well Shuttle. Immediately after nucleofection, 90uL pre-warmed cell appropriate media was added to each well, and the cells were incubated at 37°C for 15 minutes. Following incubation, cells were seeded at 1 x106 cells/mL in media supplemented with IL-2.

value: Blood

value: CRISPR KO Screen

value: IL2RA high


title: D1_Teff_Stimulated_MED11_KO_RNA

status: Public on Oct 14 2024

type: SRA

city: San Francisco

first: Alexander

last: Marson

state: CA

address: 1650 Owens St

country: USA

institute: UCSF Gladstone Institutes

library_source: transcriptomic

data_processing:
RNAseq data was processed using the pipeline described in Freimer et al 2022. In brief, fastq adapter trimming was performed with cutadapt (v2.10).
Low-quality bases were trimmed with seqtk (v0.5.0).
Reads were then aligned with STAR62 (v 2.7.10a) and mapped to GRCh38.
UMI counting and deduplication was performed with umi_tools (v1.0.1) and gene counts were generated from the deduplicated reads using featureCounts (subread v2.0.1) using Gencode v41 basic transcriptome annotation.
Quality control metrics were generated for each sample with Fastqc (v0.11.9), rseqc (v3.0.1), and Multiqc (v1.9).
Assembly: GRCh38
Supplementary files format and content: deduplicated count matrix for all files

molecule: polyA RNA

organism: Homo sapiens

source_name: Lymphocyte

growth_protocol: CD4+ regulatory and effector T cells were isolated from fresh Peripheral Blood Leukopaks (STEMCELL Technologies, #70500) from healthy human donors, after institutional review board–approved informed written consent (STEMCELL Technologies). The contents of the Leukopaks were washed twice with a 1X volume of EasySep buffer (DPBS, 2% fetal Bovine Serum (FBS), 1mM pH 8.0 EDTA) using centrifugation. The washed cells were resuspended at 200E6 cells/mL in EasySep buffer and isolated with the EasySep™ Human CD4+CD127lowCD25+ Regulatory T Cell Isolation Kit (STEMCELL Technologies, #18063), according to the manufacturer’s protocol. Following isolation with the kit, Tregs were stained Alexa Fluor® 647 anti-human CD25 Antibody (Biolegend, #302618), PE anti-Human CD127 (Beckon Dickinson, #557938), and Pacific Blue™ anti-human CD4 Antibody (Biolegend, #344620) and isolated using FACS to ensure a pure population without contaminating effector cells. After sorting pure CD4+CD127lowCD25+ Regulatory T Cells, the cells were seeded at 1x106 cells/mL in XVIVO-15 (Lonza, #02-053Q) supplemented with 55 uM 2-mercaptoethanol, 4 mM N-acetyl L-cysteine, and 200 U/mL IL-2 (Amerisource Bergen, #10101641). Teffs were seeded at 1x106 cells/mL in RPMI-1640 supplemented with 10% FCS, 2 mM L-Glutamine (Fisher Scientific #25030081), 10 mM HEPES (Sigma, #H0887-100ML), 1X MEM Non-essential Amino Acids (Fisher, #11140050), 1 mM Sodium Pyruvate (Fisher Scientific #11360070), 100 U/mL Penicillin-Streptomycin (Sigma, #P4333-100ML), and 50 U/mL IL-2 (Amerisource Bergen, #10101641). Both cell subsets were then stimulated with ImmunoCult™ Human CD3/CD28/CD2 T Cell Activator (STEMCELL Technologies, #10990) at 25 uL/mL for Tregs and 6.25 uL/mL for Teff. Following activation and electroporation, cells were split 1:2 every 48 hours to maintain an approximate density of 1x106 cells/mL and supplemented with respective doses of IL-2.

extract_protocol:
8 and 10 days after editing, respectively, resting and 48-hour restimulated cells were pelleted and resuspended at 1x106 cells per 300 µL of RNA lysis buffer (Zymo, #R1060-1-100). Cells were pipette mixed and vortexed to lyse and frozen at -80 until RNA isolation was performed. RNA was isolated using the Zymo-Quick RNA micro prep kit (#R1051) according to the manufacturer’s protocol with the following modifications: After thawing the samples, each sample was vortexed vigorously to ensure total lysis prior to loading into the extraction columns. The optional kit provided DNAse step was skipped, and instead RNA was eluted from the isolation column after the recommended washes and digested with Turbo-DNAse (Fisher Scientific, AM2238) at 37 C for 20 minutes. Following digestion, RNA was purified using the RNA Clean & Concentrator-5 kit (Zymo, #R1016) according to the manufacturer’s protocol.
The purified RNA was submitted to the UC Davis DNA Technologies and Expression Analysis Core to generate 3′ Tag-seq libraries with unique molecular indices (UMIs). Barcoded sequencing libraries were prepared using the QuantSeq FWD kit (Lexogen) for multiplexed sequencing.

treatment_protocol: Guide loaded Cas9 RNPs were assembled with custom crRNAs (Dharmacon) which were resuspended in IDT duplex buffer (IDT, #11-01-03-01) at 160uM. Dharmacon Edit-R CRISPR-Cas9 Synthetic tracrRNA (Dharmacon, #U-002005-20) also resuspended in Nuclease Free Duplex Buffer at 160uM was combined at a 1:1 molar ratio in a 96 well plate and incubated at 37°C for 30 minutes. Single-stranded donor oligonucleotides (ssODN; sequence: TTAGCTCTGTTTACGTCCCAGCGGGCATGAGAGTAACAAGAGGGTGTGGTAATATTACGGTACCGAGCACTATCGATACAATATGTGTCATACGGACACG, 100uM stock) was added to the complex at a 1:1 molar ratio and incubated at 37°C for 5 minutes. Finally, Cas9 protein (MacroLab, Berkeley, 40 µM stock) was added at a 1:2 molar ratio and incubated at 37°C for 15 minutes. The resulting RNPs were frozen at -80°C until the day of electroporation and were thawed to room temperature prior to use. 48 hours following T cell activation, the cells were pelleted at 100x g for 10 minutes and resuspended in room temperature P3 Primary Cell Nucleofector Buffer (Lonza, catalog no. V4XP-3032) at 1.5E6 cells per 17.8 µL. 1.5E6 cells were transferred to each RNP containing well and mixed gently. 25 µL of the combined RNP cell solution was transferred to a 96-well electroporation cuvette plate (Lonza, #VVPA-1002) and nucleofected with pulse code DS-137. Immediately following electroporation, the cells were gently resuspended in 90 µL warmed media and incubated at 37 C for 15 minutes. After recovery, the cells were cultured in 96 well round-bottom plates at 1x106 cells/mL for the duration of the experiment. To prevent edge effects, the guides were randomly distributed across each plate and the first and last columns and rows of each plate was filled with PBS to prevent evaporation. CRISPR-Cas9 edited cells were restimulated 48 hours prior to collection for stimulation response arrayed assays using ImmunoCult™ Human CD3/CD28/CD2 T Cell Activator (STEMCELL Technologies, #10990)

tag: tissue

value: Lymphocyte

tag: cell type

value: CD4+ Teff

tag: crispr experiment

value: CRISPR KO RNA

tag: sgrna

tag: group

tag: condition

value: Stimulated

tag: donor


title: D6_Teff_Stim_low_IL2RA_screen

library_source: genomic

molecule: genomic DNA

value: IL2RA low


title: SUM149CR, DMSO, day2, rep1

city: Boston

first: Kornelia

last: Polyak

state: MA

address: 450 Brookline Ave

department: Medical Oncology

institute: Dana-Farber Cancer Institute

data_processing:
All RNA-seq data was analyzed using the VIPER pipeline (Cornwell et al., 2018). Briefly, the STAR aligner (Dobin et al., 2013) was used to align fastq files to the human reference GRCh37/hg19 genome and produce raw gene counts. Cufflinks (Trapnell et al., 2010) was used to create normalized gene counts in terms of FPKM.
Assembly: hg19
Supplementary files format and content: CSV (comma-separated values) matrix files that include raw counts for each sample.
Supplementary files format and content: CSV (comma-separated values) matrix files that include FPKM normalized counts for each sample.

molecule: total RNA

growth_protocol: SUM149 and SUM149CR were cultured in DMEM/F12 with 5% FBS, 5 µg/mL Insulin, 1 µg/mL Hydrocortisone, and 100 µg/mL Penicillin-Streptomycin. MDAMB436 was cultured in McCoy's with 10% FBS, 10 µg/mL Insulin, and 100 µg/mL Penicillin-Streptomycin. MCF7 was cultured in DMEM/F12 with 10% FBS, 10 µg/mL Insulin, and 100 µg/mL Penicillin-Streptomycin. T47D was cultured in RPMI with 10% FBS, 10 µg/mL Insulin, and 100 µg/mL Penicillin-Streptomycin. All cells were maintained in a humidified atmosphere with 5% CO2 at 37C.

extract_protocol:
Total RNA was extracted using the Qiagen RNeasy Mini Kit (Qiagen, #74106) following the manufacturer's protocol.
RNA-seq libraries were prepared using Illumina TruSeq Stranded mRNA sample preparation kits from 500 ng of purified total RNA according to the manufacturer’s protocol. The finished dsDNA libraries were quantified by Qubit fluorometer, Agilent TapeStation 2200, and RT-qPCR using the Kapa Biosystems library quantification kit according to the manufacturer’s protocols.

treatment_protocol: Cells were seeded in 10 cm dishes in media containing 0.01% DMSO or 10 µM C70. Cells were treated for 2 or 7 days before RNA collection.

tag: cell line

value: Human breast cancer cell line

tag: genotype

value: WT

tag: treatment

value: DMSO

tag: time

value: 2 days


title: SUM149CR, C70, day7, rep3

value: 7 days


title: SUM149CR, C70, day7, rep2


title: D1_Teff_Resting_MED24_KO_RNA

value: Resting


title: Low LNP + GI.1/GII.4 rep4

city: Tianjin

first: Weiqian

last: Dai

address: Xinxing Sub-district, Heping District

country: China

institute: Tianjin Medical University

data_processing:
Illumina Casava 1.8 software used for basecalling
Raw data (raw reads) of fastp format were firstly processed through in-house perl scripts..In this step, clean data (clean reads) were obtained by removing reads containing adapter, reads containing ploy-N and low quality reads from raw data. At the same time, Q20, Q30 and GC content the clean data were calculated. All the downstream analyses were based on the clean data with high quality.
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.
featureCounts v1.5.0-p3 was used to count the reads numbers mapped to each gene.
Differential expression analysis of two conditions/groups (four biological replicates per condition) was performed using the DESeq2 R package (1.20.0).
Assembly: mm10
Supplementary files format and content: tab-delimited text files include FPKM values and gene count for each Sample
Supplementary files format and content: tab-delimited text files include FPKM values and gene count for each Sample

organism: Mus musculus

source_name: draining lymph nodes

extract_protocol:
Total RNA was isolated using TRIzol (Invitrogen) according to the manufacturer’s instructions.
RNA libraries for RNA-seq were prepared using NEBNext® Ultra™ RNA Library Prep Kit for Illumina® following manufacturer's protocols.

value: draining lymph nodes

tag: strain

value: BABL/c

value: 125μg LNPs+ 5μg GI.1/GII.4 VLP injected


title: D5_Teff_Stim_high_IL2RA_screen

value: Blood


title: Low LNP + GI.1/GII.4 rep3


title: Low LNP + GI.1/GII.4 rep2


title: D5_Teff_Stim_low_IL2RA_screen

value: CRISPR KO Screen


title: D1_Teff_Resting_MED14_KO_RNA


title: SUM149CR, C70, day7, rep1


title: D1_Teff_Resting_MED31_KO_RNA


title: D4_Teff_Stim_high_IL2RA_screen

value: IL2RA high


title: D4_Teff_Stim_low_IL2RA_screen


title: SUM149CR, C70, day2, rep2


title: Low LNP + GI.1/GII.4 rep1


title: SUM149CR, C70, day2, rep1


title: High LNP rep3

value: 1000μg LNPs injected


title: D1_Teff_Resting_AAVS1_3_KO_RNA

value: Lymphocyte

value: CD4+ Teff


title: D1_Teff_Resting_MED11_KO_RNA

value: CRISPR KO RNA


title: D1_Teff_Resting_MED30_KO_RNA


title: D2_Treg_Rest_high_IL2RA_screen

value: CD4+ Treg


title: SUM149, ZBTB7AKO, DMSO, H3K4me3_IP, rep2

data_processing:
All ChIP-seq data was analyzed using the Containerized Bioinformatics workflow for Reproducible ChIP/ATAC-seq Analysis (CoBRA) package (Qiu et al., 2021). Samples were analyzed in duplicates, and each sample was normalized to a 1% input. Reads were aligned to the hg19 genome using the bwa aligner. Peaks were called using MACS2. Differential peaks were identified using DESeq2 with padj<0.05 as the cutoff.
Assembly: hg19
Supplementary files format and content: bigWig
Supplementary files format and content: narrowPeak (except for Input samples)

growth_protocol: All SUM149 derivatives were cultured in DMEM/F12 with 5% FBS, 5 µg/mL Insulin, 1 µg/mL Hydrocortisone, and 100 µg/mL Penicillin-Streptomycin. Cells were maintained in a humidified atmosphere with 5% CO2 at 37C.

extract_protocol:
Cells were directly fixed in 15 cm cell culture plates. For ChIP-seq of histone modifications (H3K4me3), samples were crosslinked in fixing buffer (50 mM HEPES-NaOH pH 7.5, 100 mM NaCl, 1 mM EDTA) containing 1% PFA (Electron Microscopy Sciences, 15714) for 10 minutes at room temperature (i.e., single fixation). For ChIP-seq of chromatin binding proteins (KDM5A, KDM5B, ZBTB7A), samples were first crosslinked in PBS containing 2 mM DSG (Fisher Scientific 20593) for 30 minutes at room temperature. DSG was then removed, and the samples were further crosslinked with fixing buffer (50 mM HEPES-NaOH pH 7.5, 100 mM NaCl, 1 mM EDTA) containing 1% PFA for 10 minutes at 37 oC (i.e., double fixation). After PFA fixation, all samples were quenched with glycine at a final concentration of 0.125 M for 5 minutes at room temperature. Cells were washed and harvested in ice cold PBS. Nuclei were extracted by first resuspending the cells in lysis buffer for 10 minutes at 4C (50 mM HEPES-NaOH pH 8, 140 mM NaCl, 1 mM EDTA, 10% Glycerol, 0.5% IGEPAL CA-630, 0.25% Triton X-100), 1 mL of lysis buffer per 5 million cells. Nuclei were pelleted and washed once in wash buffer (10 mM Tris-HCl pH 8, 200 mM NaCl, 1 mM EDTA), 1 mL of wash buffer per 10 million (histone modification ChIP) or 25 million cells (chromatin modification ChIP). The nuclei were then pelleted and resuspended in 1 mL of shearing buffer (10 mM Tris-HCl pH 8, 1 mM EDTA, 0.1% SDS), containing 10 million (histone modification ChIP) or 25 million cells (chromatin modification ChIP). Samples were transferred to 1 mL AFA Fiber tubes (Covaris 520130), and sonicated in a Covaris E220 sonicator with the following settings: Peak Incident Power 150, Duty Cycles 5%, cycles per Burst 200. Histone modification ChIP samples were sonicated for 900 seconds. Chromatin binding protein ChIP samples were sonicated for 1,200 seconds. After sonication, Triton X-100 and NaCl were added to a final concentration of 1% Triton X-100 and 150 mM NaCl. The samples were then pre-cleared with 50 µL of Dynabeads Protein G (Fisher Scientific, 10004D) for 1 hour at 4 oC. For histone modification ChIP, primary antibodies were added to 1 mL of pre-cleared chromatin from ~10 million cells (H3K4me3 = Diagenode C154100003 at 5 µg/mL) and incubated at 4 oC overnight. For chromatin binding protein ChIP, pre-cleared samples were aliquoted and volumes adjusted using shearing buffer containing 1% Triton X-100 and 150 mM NaCl so that 1 mL contained pre-cleared chromatin from ~10 million cells. Primary antibodies were added to each aliquot and incubated  at 4 oC overnight (ZBTB7A = SantaCruz sc-33683X at 5 µg/mL, KDM5A = Abcam ab70892 at 4 µg/mL, KDM5B = Novus/sdix 22260002 at 5 µg/mL). After overnight immunoprecipitation, crosslinked complexes were precipitated with  Dynabeads G (Fisher Scientific, 10004D) for 2 hours at 4 oC. The beads were then washed once with low salt wash buffer (20 mM Tris-HCl pH 8, 150 mM NaCl, 0.1% SDS, 1% Triton-X 100, 2 mM EDTA) for 5 minutes at 4 oC, once with high salt wash buffer (20 mM Tris-HCl pH 8, 500 mM Nacl, 0.1% SDS, 1% Triton-X 100, 2 mM EDTA) for 5 minutes at 4 oC, once with wash buffer (10 mM Tris-HCl pH 8, 250 mM LiCl, 1% IGEPAL CA-630, 1% Sodium Deoxycholate, 1 mM EDTA) for 5 minutes at 4 oC, and once with TE buffer (10 mM Tris-HCl pH8, 1 mM EDTA) quickly at room temperature. DNA was eluted from the beads in 1% SDS, 100 mM NaHCO3 for 30 minutes at room temperature. Crosslinks were reversed at 65 oC overnight. RNA and protein were digested with 0.2 mg/mL RNase A for 30 minutes at 37 oC followed by 0.2 mg/mL Proteinase K for 1 hour at 55 oC. DNA was extracted with 300 µl of phenol/chloroform at pH8 and centrifuged at 20,000g for 5 min at RT. DNA in the upper layer was then precipitated with 300 µL isopropanol, 100 µL of 2 M sodium perchlorate, and 5 µl glycogen and centrifuged at 20,000g for 10 min at RT. Pelleted DNA was washed twice with 70% ethanol and resuspended in 20 µl low TE.
Libraries were prepared with the automated Swift 2S ligation chemistry and sequenced to 40M 50 bp reads pairs.

treatment_protocol: Cells were seeded in 15 cm dishes. For treatments, cells were seeded in media containing 0.01% DMSO or 10 µM C70 and treated for 7 days before collection of genomic DNA.

value: Human breast cancer cell line

tag: chip antibody

value: H3K4me3 (Diagenode, C15410003)


title: High LNP rep4


title: High LNP rep2


title: D2_Treg_Rest_low_IL2RA_screen

value: IL2RA low


title: High LNP rep1


title: D1_Teff_Resting_MED12_KO_RNA


title: SUM149, ZBTB7AKO, DMSO, KDM5B_IP, rep2

value: DMSO


title: D1_Treg_Rest_high_IL2RA_screen


title: SUM149, ZBTB7AKO, DMSO, KDM5A_IP, rep2

value: KDM5A (Abcam, ab70892)


title: D1_Treg_Rest_low_IL2RA_screen

value: Resting


title: SUM149, ZBTB7AKO, DMSO, H3K4me3_IP, rep1


title: D1_Teff_Resting_AAVS1_1_KO_RNA


title: Alum rep4

value: draining lymph nodes

value: 250μg Alum injected


title: D3_Treg_Stimulated_MED24_KO_RNA

value: Stimulated


title: SUM149CR, C70, day2, rep3

value: WT

value: 2 days


title: Alum rep3

value: BABL/c


title: Input REP3

city: Wageningen

first: Kai

last: Thoris

address: Droevendaalsesteeg 1

country: Netherlands

institute: WUR

data_processing:
Read trimming with Trim Galore!
Read mapping with HISAT2
Merging files with MergeSamFiles
Peakcalling with MACS2
Annotating peaks with ChIPseeker
Differential peak analysis with MACS2 bdgdiff
Assembly: TAIR10
Supplementary files format and content: Narrow peaks representing peaks for p<0,05
Supplementary files format and content: Bedgraphs representing normalized read files
Library strategy: DAP-seq

organism: Arabidopsis thaliana

source_name: Leaves from seedlings

growth_protocol: 2-weeks old seedlings of Arabidopsis thaliana

extract_protocol:
Recombinant proteins were produced with the TnT SP6 protein expression system from Promega, while the genomic DNA was extracted with a CTAB protocol from 2 week old Arabidopsis seedlings.
The libraries were constructed with the Truseq Illumina kit and unique adaptors were used for each sample. 400ng of DNA library was used per sample. Libraries were amplified using 20 cycles on the thermocycler. Post-amplification libraries were size selected at 250-650bp in length using Agencourt AMPure XP beads from Beckman Coulter

value: Leaves from seedlings

tag: protein epitope_tags


title: SUM149, ZBTB7AKO, DMSO, KDM5B_IP, rep1


title: D3_Treg_Stimulated_MED14_KO_RNA

value: Lymphocyte


title: SUM149, ZBTB7AKO, DMSO, KDM5A_IP, rep1


title: Input REP2


title: Alum rep2


title: D3_Treg_Stimulated_AAVS1_3_KO_RNA


title: Alum rep1


title: Naive rep4

value: nothing injected


title: Input REP1

description:
processed data file (bigwig) for three replicates combined
input_normalized_3_repl.bigwig


title: FUL-SOC1 REP3

description:
Narrow_peaks_FUL-SOC1_3repl_p_0.05.bed
FUL-SOC1_normalized_3_repl.bigwig


title: FUL-SOC1 REP2


title: Naive rep3


title: Naive rep2


title: D3_Treg_Stimulated_MED11_KO_RNA

value: CRISPR KO RNA


title: SUM149, KDM5BKO, DMSO, H3K4me3_IP, rep2


title: SUM149, KDM5BKO, DMSO, KDM5A_IP, rep2


title: D3_Treg_Stimulated_MED30_KO_RNA

value: CD4+ Treg


title: FUL-SOC1 REP1

description:
processed data files (narrow peaks and bigwig) for three replicates combined
Narrow_peaks_FUL-SOC1_3repl_p_0.05.bed
FUL-SOC1_normalized_3_repl.bigwig


title: SUM149, KDM5BKO, DMSO, ZBTB7A _IP, rep1

value: Human breast cancer cell line

value: ZBTB7A (SantaCruz, sc-33683 X)


title: Naive rep1


title: D3_Treg_Stimulated_MED12_KO_RNA


title: D3_Treg_Stimulated_AAVS1_1_KO_RNA


title: SUM149, KDM5BKO, DMSO, H3K4me3_IP, rep1

value: H3K4me3 (Diagenode, C15410003)


title: FUL-SEP3 REP3

description:
Narrow_peaks_FUL-SEP3_3repl_p_0.05.bed
FUL-SEP3_normalized_3_repl.bigwig


title: FUL-SEP3 REP2


title: SUM149, KDM5BKO, DMSO, KDM5A_IP, rep1

value: DMSO


title: D2_Treg_Stimulated_MED24_KO_RNA


title: SUM149, KDM5BKO, DMSO, ZBTB7A _IP, rep2


title: D3_Treg_Stimulated_MED31_KO_RNA


title: D2_Treg_Stimulated_MED14_KO_RNA


title: FUL-AG-SEP3 REP3

description:
Narrow_peaks_FUL-AG-SEP3_3repl_p_0.05.bed
FUL-AG-SEP3_normalized_3_repl.bigwig


title: D2_Treg_Stimulated_MED31_KO_RNA


title: FUL-AG-SEP3 REP2


title: ImPSC-1 cells, Egfp OE (overexpression), replicate 3

city: New York

first: Mara

middle: H

last: Sherman

state: NY

department: Cancer Biology and Genetics

institute: Memorial Sloan Kettering Cancer Center

data_processing:
Sequence reads were trimmed to remove possible adapter sequences and nucleotides with poor quality. The trimmed reads were mapped to the reference genome GRCm38.91 (mm10) available on ENSEMBL using the STAR aligner v.2.5.2b. The STAR aligner is a splice aligner that detects splice junctions and incorporates them to help align the entire read sequences. Unique gene hit counts were calculated by using feature Counts from the Subread package v.1.5.2. Only unique reads that fell within exon regions were counted. The gene hit counts table was used for downstream differential expression analysis. Using DESeq2, a comparison of gene expression between the groups of samples was performed. The Wald test was used to generate p-values and Log2 fold changes. Genes with adjusted p-values < 0.05 and absolute log2 fold changes > 1 were called as differentially expressed genes for each comparison.
Assembly: GRCm38.91 (mm10)
Supplementary files format and content: .txt file containing raw counts for raw counts for all sample
Supplementary files format and content: .txt file containing raw counts for raw counts for each sample

growth_protocol: Stable cells were maintained in a humidified atmosphere at 37°C with 5% CO2 and routinely passed in DMEM containing 10% FBS, 1 mM sodium pyruvate, and 1% Antibiotics-Antimycotic.

extract_protocol:
Total RNA was isolated using RNeasy Microkit per manufacturer’s instructions and quantified using NanoDrop microvolume spectrophotometer before submission for bulk RNA-sequencing.
Total RNA underwent polyA selection and RNA sequencing libraries preparation using the NEBNext Ultra II RNA Library Prep Kit for Illumina using manufacturer’s instructions (NEB, Ipswich, MA, USA)

value: Pancreatic mesenchymal cells

value: Egfp overexpression


title: ImPSC-1 cells, Kitl OE (overexpression), replicate 3

value: Kitl overexpression


title: ImPSC-1 cells, Kitl OE (overexpression), replicate 1


title: ImPSC-1 cells, Kitl OE (overexpression), replicate 2


title: SUM149, KDM5AKO, DMSO, ZBTB7A _IP, rep2


title: SUM149, KDM5AKO, DMSO, H3K4me3_IP, rep2


title: SUM149, KDM5AKO, DMSO, KDM5B_IP, rep2


title: SUM149, KDM5AKO, DMSO, ZBTB7A _IP, rep1


title: D2_Treg_Stimulated_MED11_KO_RNA


title: SUM149, KDM5AKO, DMSO, H3K4me3_IP, rep1


title: ImPSC-1 cells, Egfp OE (overexpression), replicate 2


title: ImPSC-1 cells, Egfp OE (overexpression), replicate 1


title: D2_Treg_Stimulated_MED30_KO_RNA


title: FUL-AG-SEP3 REP1

description:
processed data files (narrow peaks and bigwig) for three replicates combined
Narrow_peaks_FUL-AG-SEP3_3repl_p_0.05.bed
FUL-AG-SEP3_normalized_3_repl.bigwig


title: ImPSC-1 cells, shKitl replicate 3

value: Kitl knock down


title: FUL-AG REP2

description:
Narrow_peaks_FUL-AG_3repl_p_0.05.bed
FUL-AG_normalized_3_repl.bigwig


title: ImPSC-1 cells, shKitl replicate 2


title: FUL-SEP3 REP1

description:
processed data files (narrow peaks and bigwig) for three replicates combined
Narrow_peaks_FUL-SEP3_3repl_p_0.05.bed
FUL-SEP3_normalized_3_repl.bigwig


title: SUM149, KDM5AKO, DMSO, KDM5B_IP, rep1


title: D2_Treg_Stimulated_MED12_KO_RNA

value: Stimulated


title: SUM149, ROSA26KO, 10 µM C70, ZBTB7A _IP, rep2


title: D2_Treg_Stimulated_AAVS1_1_KO_RNA


title: ImPSC-1 cells, shKitl replicate 1


title: FUL REP3

description:
Narrow_peaks_FUL_3repl_p_0.05.bed
FUL_normalized_3_repl.bigwig


title: ImPSC-1 cells, shCtrl, replicate 3

value: WT


title: SUM149, ROSA26KO, 10 µM C70, ZBTB7A _IP, rep1


title: D1_Treg_Stimulated_MED24_KO_RNA


title: D2_Treg_Stimulated_AAVS1_3_KO_RNA


title: FUL-AG REP3


title: D1_Treg_Stimulated_MED14_KO_RNA


title: FUL REP1

description:
processed data files (narrow peaks and bigwig) for three replicates combined
Narrow_peaks_FUL_3repl_p_0.05.bed
FUL_normalized_3_repl.bigwig


title: FUL REP2


title: ImPSC-1 cells, shCtrl, replicate 1


title: D3_Teff_Rest_high_IL2RA_screen

value: Blood

value: CD4+ Teff

value: CRISPR KO Screen

value: IL2RA high

value: Resting


title: SUM149, ROSA26KO, 10 µM C70, H3K4me3_IP, rep1


title: ImPSC-1 cells, shCtrl, replicate 2


title: SUM149, ROSA26KO, 10 µM C70, H3K4me3_IP, rep2


title: D3_Teff_Rest_low_IL2RA_screen

value: IL2RA low


title: Single nuclei form uninjured heart tissue, Un4

city: Los Angeles

first: Feiyang

last: Ma

address: 610, Charles Young Dr East, TLSB 3000C

department: Molecular Biology Institute

institute: UCLA

library_source: transcriptomic single cell

data_processing:
Preprocessing of the raw data was conducted following the Cell Ranger pipeline (10X Genomics). The Cellranger output expression matrices were merged for each sample. The R package Seurat (v4.3.0) was used to cluster the cells in the merged matrix.
Assembly: mm10
Supplementary files format and content: Expression matrices

source_name: Mus uninjured heart

extract_protocol:
Mice hearts were harvest at 7 days after MI. The injured heart tissue was dissected and homogenized using nuclei lysis buffer. Nuclei pellet was resuspended and filtered through 100μm and 20μm Cell Strainer (43-50000-98, PluriStrainer). Nuclei concentrations were counted using Countess II Automated Cell Counter (Thermo Fisher Scientific).
Single nuclei gene expression libraries were created using Chromium Next GEM Single ’ell 3' (v3.1 Chemistry) (PN-1000121, 10x Genomics), Chromium Next GEM Chip G Single Cell Kit (PN-1000127, 10x Genomics), and Single Index Kit T Set A (PN-1000213, 10x Genomics) according to the manufacturer’s instructions. Briefly, nuclei were loaded to target 10,000 nuclei to form GEMs and barcode individual nuclei into cDNA libraries. Library quality was assessed using 4200 TapeStation System and D1000 ScreenTape (Agilent) and Qubit 2.0 (Invitrogen) for concentration and size distribution.

value: Mus uninjured heart

value: heart cells

value: humanized ENPP1 C57BL/6NT mice


title: D1_Treg_Stimulated_MED11_KO_RNA

value: Lymphocyte

value: CD4+ Treg

value: CRISPR KO RNA


title: D2_Teff_Rest_high_IL2RA_screen


title: FUL-AG REP1

description:
processed data files (narrow peaks and bigwig) for three replicates combined
Narrow_peaks_FUL-AG_3repl_p_0.05.bed
FUL-AG_normalized_3_repl.bigwig

value: Leaves from seedlings


title: D1_Treg_Stimulated_AAVS1_3_KO_RNA


title: SUM149, ROSA26KO, 10 µM C70, KDM5B_IP, rep1

value: Human breast cancer cell line


title: Single nuclei form uninjured heart tissue, Un3


title: D1_Treg_Stimulated_MED30_KO_RNA


title: Single nuclei form uninjured heart tissue, Un2


title: D2_Teff_Rest_low_IL2RA_screen


title: SUM149, ROSA26KO, 10 µM C70, KDM5B_IP, rep2


title: SUM149, ROSA26KO, 10 µM C70, KDM5A_IP, rep2

value: KDM5A (Abcam, ab70892)


title: SUM149, ROSA26KO, 10 µM C70, KDM5A_IP, rep1


title: Single nuclei form uninjured heart tissue, Un1


title: D1_Teff_Rest_high_IL2RA_screen


title: D1_Treg_Stimulated_MED12_KO_RNA

value: Stimulated


title: day 0.5 post-coitum (dpc) - Ccna2 cKO - replicate 4

city: Kansas City

first: Sumedha

middle: S

last: Gunewardena

state: Kansas

address: 2146 West 39th Avenue

department: Dept. of Molecular and Integrative Physiology

institute: University of kansas medical center

data_processing:
The read quality was assessed using the FastQC software.
The reads were mapped to the mouse genome (Mus musculus GRCm39; Ensembl release 105) using the STAR software.
Transcript abundance estimates were calculated using the featureCounts software.
Assembly: GRCm39
Supplementary files format and content: tab delimited text file containing raw read counts

source_name: uterus

extract_protocol:
Trizol extraction and RNA purification
RNA libraries were prepared for sequencing using standard Illumina protocols

treatment_protocol: Female mice of both genotypes were mated with known fertile wild-type (C57BL/6) mice and checked in morning at 0800h for the presence of a vaginal plug which was considered day 0.5 of pregnancy (dpc 0.5).

value: uterus

value: Ccna2 d/d

tag: Sex

value: Female

value: Ccna2 cKO


title: D1_Treg_Stimulated_MED31_KO_RNA


title: D1_Teff_Rest_low_IL2RA_screen

value: Blood

value: CD4+ Teff

value: CRISPR KO Screen


title: SUM149, ROSA26KO, DMSO, ZBTB7A _IP, rep2

value: DMSO

value: ZBTB7A (SantaCruz, sc-33683 X)


title: SUM149, ROSA26KO, DMSO, ZBTB7A _IP, rep1


title: D1_Treg_Stimulated_AAVS1_1_KO_RNA


title: D3_Treg_Resting_MED24_KO_RNA

value: Resting


title: SUM149, ROSA26KO, DMSO, H3K4me3_IP, rep2

value: H3K4me3 (Diagenode, C15410003)


title: day 0.5 post-coitum (dpc) - Ccna2 cKO - replicate 2


title: day 0.5 post-coitum (dpc) - Ccna2 cKO - replicate 3


title: D3_Treg_Resting_MED14_KO_RNA

value: Lymphocyte

value: CD4+ Treg


title: T47D, ZBTB7A_KO, DMSO, day7, rep2

value: 7 days


title: T47D, ZBTB7A_KO, C70, day7, rep2

value: Human breast cancer cell line


title: SUM149, ROSA26KO, DMSO, KDM5B_IP, rep2


title: day 0.5 post-coitum (dpc) - Ccna2 cKO - replicate 1


title: T47D, ROSA26_KO, C70, day7, rep2


title: day 0.5 post-coitum (dpc) - control - replicate 4

value: Ccna2 fl/fl

value: Control


title: SUM149, ROSA26KO, DMSO, KDM5B_IP, rep1


title: D3_Treg_Resting_MED11_KO_RNA

value: CRISPR KO RNA


title: D3_Treg_Resting_AAVS1_3_KO_RNA


title: day 0.5 post-coitum (dpc) - control - replicate 3

value: uterus


title: T47D, ZBTB7A_KO, C70, day7, rep1


title: SUM149, ROSA26KO, DMSO, KDM5A_IP, rep2

value: KDM5A (Abcam, ab70892)


title: D3_Treg_Resting_MED30_KO_RNA


title: T47D, ZBTB7A_KO, DMSO, day7, rep1


title: SUM149, ROSA26KO, DMSO, KDM5A_IP, rep1


title: D3_Treg_Resting_MED12_KO_RNA


title: SUM149, ROSA26_KO, DMSO, Input_single_fixation

description: Input for single fixed samples: H3K4me3 IPs


title: T47D, ROSA26_KO, C70, day7, rep1


title: D3_Treg_Resting_AAVS1_1_KO_RNA


title: day 0.5 post-coitum (dpc) - control - replicate 1

value: Female


title: T47D, ROSA26_KO, DMSO, day7, rep1


title: ret/wt, biol rep 6, db

city: Lausanne

first: Tania

last: Wyss

address: Rue du Bugnon 25A

country: Switzerland

institute: SIB Swiss Institute of Bioinformatics

data_processing:
Libraries were sequenced using the NovaSeq 6000 Control Software (v. 1.6.0) and RTA (base-calling, v. 3.4.4). Demultiplexing and fastq generation were completed using bcl2fastq (v. 2.20.0.422).
Barcode processing and single-nucleus calling were performed using the Cell Ranger analysis pipeline (v. 5.0.1,10x Genomics). Cellranger count was used with the --include-introns parameter to align sequencing reads of each sample to the mm10 reference genome (version refdata-gex-mm10-2020-A downloaded from the 10x Genomics website)
Assembly: mm10 (refdata-gex-mm10-2020-A, which corresponds to Ensembl release 98, and GENCODE M23)
Supplementary files format and content: For each library of single nuclei, we provide the 3 files generated by the Cell Ranger v. 5.0.1 pipeline within the filtered_feature_bc_matrix folder (i.e the nuclei called by Cell Ranger): barcodes.tsv.gz (tab-separated file, contains the nucleus barcodes),  features.tsv.gz (tab-separated file, contains the genes included in the mm10 transcriptome) and matrix.mtx.gz (sparse matrix format, contains the raw counts per gene (in the rows), per nucleus (in the columns)). The prefix of the three files per library correspond to the sample description.

molecule: nuclear RNA

source_name: brain: deep brain regions

growth_protocol: Mice were housed in individually ventilated type 2L cages under optimized hygienic conditions. The cages were maintained in a temperature and humidity regulated environment, enriched with standard bedding material, red house, tissues, and paper crinkles, and housed in a room under 12/12 h light/dark cycle.

extract_protocol:
Tissue was processed for single nuclei isolation as previously described in Zhou et al. (Zhou et al., 2020).
Isolated mouse nuclei were subjected to droplet-based 5′ end massively parallel single-cell RNA sequencing using Chromium Single Cell 5′ Reagent Kits per the manufacturer’s instructions (10x Genomics). Library type = 10x 5'GEX V2, Dual Index

treatment_protocol: Six female Pdgfbret/ret mice and six Pdgfbret/wt mice (5 females, 1 male) aged 5 months were used for single nuclei RNA sequencing analysis. Mice were deeply anesthetized with an overdose of Xyalzine/Ketamine in saline and perfused with cold PBS containing 10 units/mL of Heparin. The brains were macro-dissected on ice into cortical (non-calcification prone) and deep brain (calcification prone) regions and frozen on dry ice.

value: brain:deep brain regions

value: nuclei

value: Pdgfbret/wt

tag: age

value: 4m 7d

value: male


title: D3_Treg_Resting_MED31_KO_RNA

value: Resting


title: D2_Treg_Resting_MED24_KO_RNA


title: T47D, ROSA26_KO, DMSO, day7, rep2

value: DMSO


title: HEK Blue hTLR4 cells, LPS S. minnesota 2

city: Bonn

first: Felicitas

last: Lauber

address: Gerhard-Domagk-Str. 3

department: Pharmaceutical Institute

country: Germany

institute: University of Bonn

description:
HEKBluehTLR4_GroupAvgEXP_StatPara.xlsx
HEKBluehTLR4_processedData_LPS.SM...Control.xlsx

data_processing:
Preprocessing was performed using nf-core rnaseq pipeline v 3.10.1
Sequence reads were trimmed for adaptor sequence and quality sequence using TrimGalore v 0.6.7
Alignment of the trimmed reads against human genime (GRCh38) was performed using STAR v 2.7.9a
Aligned data were used as input for Salmon v 1.9.0 which employs pseudoalignment to estimate transcript abundances
Assembly: GRCh38
Supplementary files format and content: excel files include raw counts for each sample
Supplementary files format and content: excel files include comparison of samples and gene names

source_name: HEK Blue hTLR4 cells

growth_protocol: HEK Blue hTLR4 reporter cells were culutred in high glucose DMEM containing 10% FBS, 2 mM L-Glutamine and antibiotics (Penicillin/Streptomycin/Normocin/Selection) in humidified atmosphere with 5% CO2 at 37 °C.

extract_protocol:
RNA was harvested using innuPREP RNA Mini Kit 2.0 (Analytik Jena)
RNA libaries for RNA-seq were prepared using QuantSeq 3'-mRNA Library Prep Kit (Lexogen).

treatment_protocol: HEK Blue hTLR4 reporter cells were incubated with medium with or without 10 ng/ml of LPS E. coli or LPS S. minnesota for 3 h.

value: HEK Blue hTLR4 cells

value: Human embryonic kidney cells

value: LPS S. minnesota 10 ng/ml


title: D2_Treg_Resting_MED14_KO_RNA

value: Lymphocyte

value: CD4+ Treg


title: MCF7, ZBTB7A_KO, C70, day7, rep2

value: Human breast cancer cell line

value: 7 days


title: ret/wt, biol rep 6, ctx

source_name: brain: cortical regions

value: brain:cortical regions


title: ret/ret, biol rep 6, db

value: Pdgfbret/ret

value: 4m 9d

value: female


title: MCF7, ZBTB7A_KO, DMSO, day7, rep2


title: SUM149, ROSA26KO, DMSO, H3K4me3_IP, rep1

value: H3K4me3 (Diagenode, C15410003)


title: MCF7, ROSA26_KO, C70, day7, rep2


title: HEK Blue hTLR4 cells, LPS S. minnesota 1


title: D2_Treg_Resting_MED31_KO_RNA

value: CRISPR KO RNA


title: D2_Treg_Resting_AAVS1_3_KO_RNA


title: HEK Blue hTLR4 cells, LPS E. coli 2

description:
HEKBluehTLR4_GroupAvgEXP_StatPara.xlsx
HEKBluehTLR4_processedData_LPS.EC...Control.xlsx

value: LPS E. coli 10 ng/ml


title: ret/ret, biol rep 6, ctx

value: nuclei


title: MCF7, ROSA26_KO, DMSO, day7, rep2


title: day 0.5 post-coitum (dpc) - control - replicate 2

value: uterus

value: Ccna2 fl/fl

value: Female

value: Control


title: D2_Treg_Resting_MED11_KO_RNA

value: Resting


title: HEK Blue hTLR4 cells, LPS E. coli 1

value: HEK Blue hTLR4 cells

value: Human embryonic kidney cells


title: MCF7, ZBTB7A_KO, C70, day7, rep1

value: Human breast cancer cell line


title: ret/wt, biol rep 5, ctx

value: Pdgfbret/wt


title: HEK Blue hTLR4 cells, control 2

description:
HEKBluehTLR4_GroupAvgEXP_StatPara.xlsx
HEKBluehTLR4_processedData_LPS.EC...Control.xlsx
HEKBluehTLR4_processedData_LPS.SM...Control.xlsx

value: medium


title: SUM149, ROSA26_KO, DMSO, Input_double_fixation

description: Input for double fixed samples: KDM5A, KDM5B, and ZBTB7A IPs

value: DMSO


title: D2_Treg_Resting_MED30_KO_RNA

value: Lymphocyte

value: CD4+ Treg


title: MCF7, ZBTB7A_KO, DMSO, day7, rep1

value: 7 days


title: D2_Treg_Resting_MED12_KO_RNA

value: CRISPR KO RNA


title: ret/ret, biol rep 5, db

value: brain:deep brain regions

value: Pdgfbret/ret

value: 4m 7d

value: female


title: HEK Blue hTLR4 cells, control 1


title: ret/ret, biol rep 5, ctx

value: brain:cortical regions

value: nuclei


title: MCF7, ROSA26_KO, C70, day7, rep1


title: THP-1 cells, LPS S. minnesota 2

description:
THP-1macrophages_GroupAvgEXP_StatPara.xlsx
THP-1macrophages_processedData_LPS.SM...Control.xlsx

source_name: THP-1 macrophages

growth_protocol: THP-1 macrophages were derived from THP-1 monocytes using PMA (Phorbol 12-myristate 13-acetate). The cells were cultured in RPMI containing 10% fetal bovine serum (FBS), 2 mM L-Glutamine and antibiotics (Penicillin/Streptomycin) in humidified atmosphere with 5% CO2 at 37 °C.

extract_protocol:
RNA was harvested using innuPREP RNA Mini Kit 2.0 (Analytik Jena)
RNA libaries for RNA-seq were prepared using QuantSeq 3'-mRNA Libraray Prep Kit (Lexogen).

treatment_protocol: THP-1 macrophages were incubated with medium with or without 10 ng/ml of LPS E. coli or LPS S. minnesota for 3 h.

value: THP-1 macrophages

value: macrophages

value: LPS S. minnesota 10 ng/ml


title: D2_Treg_Resting_AAVS1_1_KO_RNA

value: Resting


title: MCF7, ROSA26_KO, DMSO, day7, rep1

value: Human breast cancer cell line


title: D1_Treg_Resting_MED24_KO_RNA


title: MDAMB436, ZBTB7A_KO, C70, day7, rep2


title: THP-1 cells, LPS E. coli 2

description:
THP-1macrophages_GroupAvgEXP_StatPara.xlsx
THP-1macrophages_processedData_LPS.EC...Control.xlsx

value: LPS E. coli 10 ng/ml


title: ret/ret, biol rep 4, db


title: ret/wt, biol rep 5, db

value: Pdgfbret/wt

value: 4m 9d


title: THP-1 cells, LPS S. minnesota 1


title: THP-1 cells, LPS E. coli 1


title: ret/wt, biol rep 4, db


title: D1_Treg_Resting_MED14_KO_RNA

value: Lymphocyte

value: CD4+ Treg


title: ret/wt, biol rep 4, ctx


title: MDAMB436, ROSA26_KO, C70, day7, rep2

value: 7 days


title: D1_Treg_Resting_AAVS1_3_KO_RNA

value: CRISPR KO RNA


title: MDAMB436, ROSA26_KO, DMSO, day7, rep2

value: DMSO


title: THP-1 cells, control 2

description:
THP-1macrophages_GroupAvgEXP_StatPara.xlsx
THP-1macrophages_processedData_LPS.EC...Control.xlsx
THP-1macrophages_processedData_LPS.SM...Control.xlsx

value: medium


title: MDAMB436, ZBTB7A_KO, C70, day7, rep1


title: ret/ret, biol rep 3, ctx

value: Pdgfbret/ret

value: female


title: ret/wt, biol rep 3, db

value: brain:deep brain regions

value: nuclei


title: ret/ret, biol rep 3, db


title: CA9 NEG & HRE POS (named HRE) – replicate 3

city: Woluwé

first: Jérôme

last: Ambroise

address: Avenue Hippocrate 54

department: Institut de Recherche Expérimentale et Clinique

country: Belgium

institute: Université catholique de Louvain

data_processing:
All sequencing data were analyzed using the Automated Reproducible MOdular workflow for preprocessing and differential analysis of RNA-seq data (ARMOR v1.5.4) pipeline.
In this pipeline, reads underwent a quality check using FastQC (Babraham Bioinformatics). Quantification and quality control results were summarized in a MultiQC report before being mapped using Salmon to the transcriptome index which was built using all Ensembl cDNA sequences obtained in the Homo_sapiens.GRCh38.cdna.all.fa file.
Assembly: Homo_sapiens.GRCh38.cdna.all.fa
Supplementary files format and content: Excel file with the matrix of raw counts.

source_name: hypopharyngeal tumor

growth_protocol: 1500 cells were seeded in ultra-low attachment (ULA) 96-well plates and grown as 3D spheroids for 7 days

extract_protocol:
Total RNA was isolated with RNeasy Spin Columns (Qiagen).
Libraries were prepared with the TruSeq Standarded mRNA Library Prep Kit (#20020594; Illumina) from 1 μg RNA

treatment_protocol: Cells were dissociated from 3D spheroids  and isolated by FACS using CA9 antibodies/Alexa 568 secondary antibody and GFP (resulting from HRE-GFP expression)

value: hypopharyngeal tumor

value: FaDu

value: cancer cell line


title: D1_Treg_Resting_MED11_KO_RNA

value: Resting


title: MDAMB436, ZBTB7A_KO, DMSO, day7, rep1

value: Human breast cancer cell line


title: CA9 NEG & HRE POS (named HRE) – replicate 2


title: D1_Treg_Resting_MED30_KO_RNA


title: MDAMB436, ZBTB7A_KO, DMSO, day7, rep2


title: MDAMB436, ROSA26_KO, C70, day7, rep1


title: ret/ret, biol rep 4, ctx

value: brain:cortical regions


title: D1_Treg_Resting_MED12_KO_RNA


title: CA9 NEG & HRE POS (named HRE) – replicate 1


title: ret/ret, biol rep 2, db


title: MDAMB436, ROSA26_KO, DMSO, day7, rep1


title: D1_Treg_Resting_AAVS1_1_KO_RNA


title: DOUBLE NEG (named NEG) – replicate 3


title: D1_Treg_Resting_MED31_KO_RNA


title: DOUBLE NEG (named NEG) – replicate 2


title: MIAPACA-2, HBS1L 96, dox, rep 2

city: South San Francisco

first: Sarah

last: Kongpachith

address: 1000 Gateway Blvd

institute: AbbVie

data_processing:
Reads were aligned using Illumina DRAGEN software.
Counts were calculated using featureCounts from subread package.
Lowly expressed counts were removed with the edgeR package (minimum count = 10).
Counts were adjusted by TMM normalization and limma-voom transformation.
Assembly: GRCh38.p13
Supplementary files format and content: .csv file containing raw counts for each sample
Supplementary files format and content: .csv file containing TMM-normalized log2 cpm values for each sample

growth_protocol: Generated dox inducible Cas9/sgRNA cells were cultured in RPMI supplemented with 10% tetracycline (tet) free FBS (Takara).

extract_protocol:
RNA was harvested using Qiagen RNeasy Midi or Mini kit.
Libraries were prepped using the NEB Ultra II RNA library prep kit.

treatment_protocol: Cells were treated with or without 2 µg/mL dox (Sigma) to induce knockout and samples collected at 48 and 96 hours.

value: 96 hours

tag: doxycycline


title: ret/ret, biol rep 2, ctx

tag: tissue


title: ret/wt, biol rep 2, db

tag: cell type

value: Pdgfbret/wt


title: DOUBLE NEG (named NEG) – replicate 1


title: Mcrs1ox,Mouse18

city: Shanghai

first: Xiaofei

last: Yu

address: 2005 Songhu Rd

institute: Fudan University

data_processing:
Raw fastq files were filtered and trimmed with fastp (v 0.22.0)
The filtered reads were aligned to GRCm38 reference genome with HISAT2 (v 2.2.1)
Raw counts were determined with featureCounts (v2.0.1).
The raw counts matrix was imported to R  (v 4.2.1). Data normalization and differential gene expression analysis were performed with the limma package.
Assembly: GRCm38
Supplementary files format and content: Table-delimited text files containing the raw counts of each sample
Supplementary files format and content: Xls files containing the differencially expression genes

source_name: Flanks bearing tumor tissue

growth_protocol: 1x10^6 tumor cells were resuspened in 100 uL DPBS and subcutaneously injected into the flanks of mice. Tumor growth was monitored every 3-4 days with a caliper and tumor tissues were harvested 4 weeks after injection.

extract_protocol:
Tumor tissues were harvested and processed with a RNAsimple total RNA kit according to the manufacturer’s instructions. The RNA integrity was examined with a Qsep 100.
Total RNA was subjected to polyA selection and library preparation with a TruSeq Stranded mRNA LT Kit.

value: Flanks bearing tumor tissue

value: HT

value: Pancreatic cancer cells


title: THP-1 cells, control 1

value: THP-1 macrophages

value: macrophages


title: Mcrs1ox,Mouse17


title: ret/wt, biol rep 3, ctx


title: DOUBLE POS (named POS) – replicate 3


title: ret/wt, biol rep 2, ctx


title: ret/wt, biol rep 1, db

value: 5m 3d


title: MIAPACA-2, HBS1L 96, no dox, rep 3


title: DOUBLE POS (named POS) – replicate 1


title: ret/wt, biol rep 1, ctx

value: female


title: DOUBLE POS (named POS) – replicate 2


title: Mcrs1ox,Mouse13


title: MIAPACA-2, HBS1L 96, dox, rep 3


title: MIAPACA-2, HBS1L 96, no dox, rep 1


title: Control,Mouse3

value: Vector Control


title: Control,Mouse4


title: ret/ret, biol rep 1, ctx

value: nuclei

value: Pdgfbret/ret


title: Mcrs1ox,Mouse24


title: MIAPACA-2, HBS1L 96, no dox, rep 2


title: CA9 POS & HRE NEG (named CA9) – replicate 3

value: hypopharyngeal tumor

value: FaDu


title: Control,Mouse1


title: Single nuclei form injured heart tissue, hENPP1mAb 3,  7 days post MI

source_name: Mus injured heart

value: Mus injured heart

value: heart cells

value: humanized ENPP1 C57BL/6NT mice


title: MIAPACA-2, PELO 96, dox, rep 3

value: 96 hours

value: PELO


title: MIAPACA-2, PELO 96, dox, rep 1


title: MIAPACA-2, HBS1L 96, dox, rep 1


title: Control,Mouse2

value: Flanks bearing tumor tissue


title: ret/ret, biol rep 1, db

value: brain:deep brain regions


title: ATAC-Seq spleen_FlcnKO5

city: Memphis

first: Hongbo

last: Chi

state: TN

address: 262 Danny Thomas Place

department: Immunology

institute: St Jude Children's Research Hospital

description: sgFlcn spleen OT-1 cells

data_processing:
Adapter sequences were removed using cutadapt v.1.9
and aligned them to the mouse genome mm10 (GRCm38_68 from Sanger;ftp://ftp-mouse.sanger.ac.uk/ref/GRCm38_68.fa) using the Burrows–Wheeler algorithm32(version 0.5.9-r26-dev, default parameter)
duplicated reads were then marked with Picard (version 1.65 (1160)) and only non-duplicated reads were kept, using samtools (parameter ‘-q 1 -F 1024’ version 0.1.18 (r982:295))
After adjustment using the Tn5 shift (by which reads were offset by +4bp for the sense strand and −5bp for the antisense strand), we separated reads into nucleosome-free, mononucleosome, dinucleosome and trinucleosome by fragment size
performed peak-calling for nucleosome-free reads using MACS2(version 2.1.0.20150603, default parameters with ‘–extsize 200 –nomodel’, merged by bedtoolsif within 100bp) for individual samples
Assembly: mm10
Supplementary files format and content: Consensus peaks and fragment counts for all samples

source_name: sgFlcn-transduced OT-1 cells purified from the spleen from the same host on day 7.5 post-LM-OVA infection

growth_protocol: sgNTC or sgFlcn-transduced OT-1 cells were sort purified from the spleen and siIEL from the same host on day 7.5 post-LM-OVA infection

extract_protocol:
Cells were lysed in 50μl ATAC-seq lysis buffer (10mM Tris-HCl, pH7.4, 10mM NaCl, 3mM MgCl2, 0.1% IGEPAL CA-630 detergent) on ice for 10min. Resulting nuclei were pelleted at 500gfor 10min at 4 °C. Supernatant was carefully removed with a pipette and discarded. The pellet was resuspended in 50μl transposase reaction mix (25μl 2×TD buffer, 22.5μl nuclease-free water, 2.5μl transposase) and incubated for 30min at 37 °C. After the reaction, the DNA was cleaned up using the Qiagen MinElute kit
The barcoding reaction was run using the NEBNext HiFi kit on the basis of the manufacturer’s instructions and amplified for five cycles


title: Single nuclei form injured heart tissue, hENPP1mAb 2,  7 days post MI


title: CA9 POS & HRE NEG (named CA9) – replicate 2

value: cancer cell line


title: MIAPACA-2, PELO 96, no dox, rep 3


title: Organoid, Patient 380, Coculture [mix_JHH380CoOrg]

city: Baltimore

first: Jacquelyn

middle: W

last: Zimmerman

state: Maryland

address: 1650 Orleans St, Room 186

institute: Johns Hopkins Sidney Kimmel Comp. Cancer Center

data_processing:
Salmon v.1.9.0
Alignment was performed utilizing Salmon v1.9.0 on the Joint High-Performance Computing Exchange (JHPCE). “salmon_partial_sa_index__default.tgz” was used as the index for alignment for the HG38 genome, which was premade and available on refgenie (http://refgenomes.databio.org/v3/genomes/splash/2230c535660fb4774114bfa966a62f823fdb6d21acf138d4). Salmon quantification, [salmon quant], was completed in mapping-based mode (--validateMappings) with automatic library detection. Salmon quantifications were the imported into R using tximport v1.20.0, with the use of the “tgMap.tsv” file also provided in the HG38 refgenie repository for accurate gene-level summarization.
We evaluated sample quality from the distribution of reads as visualized in a boxplot of log counts. We observed no samples with zero median expression, reflective of a low read count, so samples were not of low quality. We used principal component analysis (PCA) of the variance stabilization transform (vst) RNA-seq data to evaluate sample clustering. Projection of the discovered CoGAPS Pattern 7 onto the VST data was completed using ProjectR (V1.8.0). The bulk-seq normalized data and CoGAPs feature loadings were run through the projectR function of the package(14). The projection results were combined with the bulk-seq metadata and plotted using ggplot2 (V3.3.5) and paired t-test results added using ggpubr (V0.4.0)(14).
Analysis completed using DESeq2
Assembly: HG38
Supplementary files format and content: tab-separated values

source_name: pancreas

extract_protocol:
Cells that were coculture were sorted by FACS and immediately underwent RNA extraction (Qiagen RNeasy kit 74004) per manufacturers protocol. RNA was quantified at 260nm using NanoDrop1000 (Thermo Fisher Scientific) and 200ug of RNA was sent to Novogene Corporation Inc. RNA integrity (RIN) was measured at Novogene with Agilent 2100 Bioanalyzer (Agilent, Santa Clara, CA). All samples were required to demonstrate RIN > 6.8 and ranged from 7.2 to 10.0.
Messenger RNA was purified from total RNA using poly-T oligo-attached magnetic beads. After fragmentation, the first strand cDNA was synthesized using random hexamer primers, followed by the second strand cDNA synthesis using either dUTP for directional library or dTTP for non-directional library. For the non-directional library, it was ready after end repair, A-tailing, adapter ligation, size selection, amplification, and purification. For the directional library, it was ready after end repair, A-tailing, adapter ligation, size selection, USER enzyme digestion, amplification, and purification.

value: pancreas

tag: disease type

value: PDAC

value: epithelium

value: coculture


title: MIAPACA-2, PELO 96, dox, rep 2

tag: sgrna


title: MIAPACA-2, PELO 96, no dox, rep 2


title: CA9 POS & HRE NEG (named CA9) – replicate 1


title: ATAC-Seq spleen_FlcnKO4

description: sgFlcn spleen OT-1 cells

value: sgFlcn-transduced OT-1 cells purified from the spleen from the same host on day 7.5 post-LM-OVA infection


title: MIAPACA-2, PELO 96, no dox, rep 1


title: Single nuclei form injured heart tissue, IgG2,  7 days post MI

value: IgG


title: ATAC-Seq spleen_Control4

description: sgNTC spleen OT-1 cells

source_name: sgNTC-transduced OT-1 cells purified from the spleen from the same host on day 7.5 post-LM-OVA infection


title: Organoid, Patient 380, Monoculture [JHH380Org]

value: monoculture


title: Organoid and Fibroblast, Patient 3303

data_processing:
Cellranger v6.0.0
Cellranger (V6.0.0) was used to generate the feature-barcode matrices, aligned to the hg38 genome. Multiseq10x (V1.0) was used as the preprocessing pipeline companion to split the MULTI-seq FASTQs into cell barcode, unique molecular identifiers (UMI), and sample barcode sequences. Reads that did not align with >1 mismatch to any reference sequence and reads representing duplicated UMIs on a cell-by-cell basis were removed. Demultiplex (V1.0.2) was used for demultiplexing the data.
The 3DGE data were log normalized, linear dimension was reduced using principle component analysis, and differentially expressed genes were identified in Seurat by Wilcoxon Rank Sum Test (V4.0.1). Additional annotations of Moffitt classifiers, denoting classical and basal epithelial subtypes, and CAF subtypes were added to the Seurat object metadata based on the clustering13. Co-culture cell types were parsed based on these annotations and the barcode distinctions. Projection of the discovered CoGAPS Pattern 7 onto the 12HR MULTI-seq expression data was completed using ProjectR (V1.8.0). The MULTI-seq expression data and CoGAPs feature loadings were run through the projectR function of the package14. The projection results were combined with the MULTI-seq metadata and plotted using ggplot2 (V3.3.5) and Wilcoxon results added using ggpubr (V0.4.0)14
Analysis completed using Seurat
Assembly: HG38
Supplementary files format and content: PDAC_Multiseq_FinalAnnotations.csv = comma-separated values; single cell metadata
Supplementary files format and content: PDAC_Multiseq_FilteredCounts.csv: comma-separated values; single cell counts post filtering
Supplementary files format and content: PDAC_Multiseq_FinalSeuratObj.rds: R Data Serialization; single cell final seurat object used in analysis

extract_protocol:
Patients with PDAC undergoing endoscopic biopsy or surgical resection were enrolled in IRB-approved tissue acquisition protocols at Johns Hopkins Hospital and Massachusetts General Hospital (MGH) (NCT03563248). Patient-derived organoids (PDOs) were generated from patient surgical specimens following a combination of mechanical and enzymatic dissociation as previously described11. CAFs were extracted from surgical resection specimens after straining remnant tissue through a 70µm cell strainer and washed twice with human organoid wash media (Advanced DMEM/F12, 10mM HEPES, 1x GlutaMAX, 100µg/mL Primocin, 0.1% BSA) with centrifugation between washes. For co-culture, organoids were combined with patient-matched CAFs in Matrigel (Corning, 356234) at a 1:10 ratio of organoids to CAFs. In parallel, CAFs and PDOs were plated separately in Matrigel. Co-cultures and monocultures were plated in 24- well tissue culture dishes and extracted after 12 hours using Cell Recovery Solution (Corning, 354253) and incubated on ice at 4°C for 45 minutes for Matrigel depolymerization. Cells were then pelleted and washed in human organoid wash media prior to pelleting again. Organoids were dissociated to single cells using TrypLE Express (ThermoFisher Scientific, 12604013) following manufacturer instructions.
Single-cells were barcoded using the MULTI-seq protocol as previously described. Single-cell transcriptomics library prep was completed using the 10x Genomics Chromium Single Cell 3’ Gene Expression Dual Index Library (V3.1) according to manufacturer specifications. Library preparations quality were analyzed using the 2100 perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in bioRxiv preprint doi: https://doi.org/10.1101/2022.07.14.500096; this version posted October 10, 2022. The copyright holder for this Bioanalyzer (Agilent). Sequencing was completed at the Johns Hopkins Genetic Resources Core Facility (GCRF).

value: epithelium and stroma


title: MIAPACA-2, control 96, dox, rep 3

value: NTC


title: Single nuclei form injured heart tissue, IgG3,  7 days post MI

value: Mus injured heart


title: ATAC-Seq spleen_Control5

description: sgNTC spleen OT-1 cells

value: sgNTC-transduced OT-1 cells purified from the spleen from the same host on day 7.5 post-LM-OVA infection


title: Organoid, Patient 362, Monoculture [JHH362Org]


title: Organoid, Patient 362, Coculture [JHH362CoOrg]


title: ATAC-Seq spleen_FlcnKO3

description: sgFlcn spleen OT-1 cells


title: Single nuclei form injured heart tissue, IgG1,  7 days post MI

value: heart cells

value: humanized ENPP1 C57BL/6NT mice


title: ATAC-Seq spleen_Control3

description: sgNTC spleen OT-1 cells


title: Single nuclei form injured heart tissue, hENPP1mAb 1,  7 days post MI


title: ATAC-Seq spleen_FlcnKO2

description: sgFlcn spleen OT-1 cells


title: MIAPACA-2, control 96, dox, rep 1

value: 96 hours


title: Organoid, Patient 348, Monoculture [JHH348Org]


title: Tumor-infiltrating lymphocytes,TCR

library_source: other

data_processing:
Base calling using Illumina Bcl2fastq
The raw data of RNA-seq and TCR-seq were aligned to the GRCm38 reference genome using 10X cellranger(v7.1.0).
Data filtering and statistical analysis were performed with Seurat (v4.0.5).
Assembly: GRCm38
Supplementary files format and content: Cellranger mRNA output files: barcodes.tsv.gz; features.tsv.gz;matrix.mtx.gz
Supplementary files format and content: Cellranger TCR output files: TCR_all_contig_annotations.csv
Supplementary files format and content: Seurat object: Hashtagged_TIL_Mcrs1oxVSControl.Rds
Supplementary files format and content: Hashtag Information for Cellranger: hashtag_info.csv

molecule: other

source_name: Flank bearing tumor tissue

extract_protocol:
Tumor-infiltrating lymphocytes of Mcrs1ox or control tumor were isolated, blocked with TruStain FcX and stained with TCR-b (H57-597) and CD90.2 (30-H12) for 30min at 4°C. Cells were then washed twice and TCRb+ CD90.2+ cells were sorted with a FACSAria Fusion Flow Cytometer, with dead cells excluded as DAPI+. Sorted cells were stained with the Biolegend TotalSeq Hashtags and loaded into a 10x Chromium at a density of 1x 10^3 cells/uL in 0.04% BSA.
Library was prepared with a Chromium Single Cell 3’ v2 Reagent Kits (10X Genomics) following the manufacurer's instruction.

value: Flank bearing tumor tissue

value: lymphocytes

value: WT


title: MIAPACA-2, control 96, no dox, rep 3


title: ATAC-Seq spleen_Control2

description: sgNTC spleen OT-1 cells


title: Tumor-infiltrating lymphocytes,RNA


title: GE_p38i_rpl2__FBC

city: Milan

first: Raffaella

last: Di Micco

address: Via Olgettina, 60

country: Italy

institute: San Raffaele Telethon Institute for Gene Therapy

description:
10x Genomics  feature barcode (FBC)
GEX_8_barcodes.tsv.gz
GEX_8_features.tsv.gz
GEX_8_matrix.mtx.gz

data_processing:
Illumina sequencer’s base call files (BCLs) were demultiplexed, for each flow cell directory, into FASTQ files using Cellranger mkfastq with default parameters (v. 6.1.1, https://github.com/10XGenomics/cellranger)
FASTQ files with 3′ gene expression (GEX) and feature barcode (FBC) libraries were then processed using Cellranger count with default parameters.
HTO data, of each sample separately, were normalized with a centered log-ratio (CLR) transformation across features. Cell demultiplexing was performed with HTODemux function in Seurat (v 4.1.1), considering an average positive quantile of 0.9 per each sample. Finally, only singlet cells, with a unique hashtag identifying a sorted population, were retained, and collected within a unique Seurat object.
Counts were normalized using Seurat function NormalizeData with default parameters. Cell cycle scores were calculated using the CellCycleScoring function, providing as input a previously reported gene list (Nestorowa et al., 2016).
Expression data were then scaled using the ScaleData function, regressing on the cell cycle phases (S, G2M scores as calculated with CellCycleScoring function), on the percentage of mitochondrial genes and the total number of reads per cell.
Assembly: custom cellragner genome GRCh38+AAV6 with PGK-GFP cassette
Supplementary files format and content: Tab separated matrices of counts with barcodes and features info

source_name: Cord blood

extract_protocol:
24h after the editing treatment, viable CD34+ cells (negativite to ZombieAqua viable dye) were sorted according to surface expression of CD34+CD133+CD45RA-CD90+GFP+ and CD34+CD133+CD45RA-CD90+GFP-. Sorted population where stained for 30’ at 4°C with Total Seq-B hashtag antibodies (H3 for CD34+CD133+CD45RA-CD90+GFP+ GE DMSO and GE p38i; H4 for CD34+CD133+CD45RA-CD90+GFP- GE DMSO and H5 for CD34+CD133+CD45RA-CD90+GFP- GEp38i) and viable cells were counted with Trypan Blue solution 0.4% (GIBCO) and 5x104 viable cells (2,5x104 from each population) were utilized for the subsequent procedure (estimated recovery: 3x104 cells/sample). Briefly, single cells were partitioned in Gel Beads in Emulsion (GEMs) and lysed, followed by RNA barcoding, reverse transcription and PCR amplification (11 cycles).
scRNA-Seq libraries were prepared according to the manufacturer’s instructions, checked and quantified on LabChip GX Touch HT (Perkin Elmer) and Qubit 3.0 (Invitrogen) instruments.

value: Cord blood

value: HSPC

value: AAV6 with PGK-GFP cassette

tag: hashtag

value: Alive cells stained with Hashtag 1 and Hashtag 2 and HSC pool (CD45RA-CD90+GFP+) stained with Hashtag 3 and HSC pool (CD45RA-CD90+GFP-) stained with Hashtag 5


title: HT,ChIP,Mcrs1,rep2

data_processing:
Raw fastq files were filtered and trimmed with fastp (v 0.22.0)
The filtered reads were aligned to GRCm38 reference genome with Bowtie2(2.5.0)
Peak calling was performed with MACS2(2.2.7.1) with the following parameters: -f BAMPE --broad --broad-cutoff 0.001 --nomodel.
For track visualization, bigwig files were generated using deeptools(v 3.5.1) subcommand bamCoverage and normalized using FPKM.
Assembly: GRCm38
Supplementary files format and content: bigwig files for track visulization
Supplementary files format and content: BroadPeak file

source_name: HT

growth_protocol: HT cells were cultured in RPMI-1640 supplemented with 10% Fetal bovine serum and 1x Penicillin-Streptomycin.

extract_protocol:
6x10^6 cells were cross-linked with 1% formaldehyde for 15 mins and quenched with glycine. Genomic DNA was sheared by sonication with Qsonica Q800R2 sonicator for 15mins (15s on: 45s off). Input DNA were taken and stored at -80 ℃. Antibodies were first incubated with Protein A beads and Protein G beads, and the beads-antibody complex were used for immunoprecipitation. After reverse cross-linking, DNA was purified by a HiPure Gel DNA Mini Kit.
The sequencing library was prepared with VAHTS Universal DNA Library Prep Kit for Illumina V3 following the manufacturer’s manual.

value: HT

value: Pancreatic cancer cells

tag: antibody

value: MCRS1(Proteintech, 11362-1-AP)


title: ATAC-Seq spleen_FlcnKO1

description: sgFlcn spleen OT-1 cells

value: sgFlcn-transduced OT-1 cells purified from the spleen from the same host on day 7.5 post-LM-OVA infection


title: GE_p38i_rpl1__FBC

description:
10x Genomics  feature barcode (FBC)
GEX_7_barcodes.tsv.gz
GEX_7_features.tsv.gz
GEX_7_matrix.mtx.gz


title: ATAC-Seq spleen_Control1

description: sgNTC spleen OT-1 cells


title: HT,ChIP,Mcrs1,rep1


title: GE_DMSO_rpl2__FBC

description:
10x Genomics  feature barcode (FBC)
GEX_6_barcodes.tsv.gz
GEX_6_features.tsv.gz
GEX_6_matrix.mtx.gz

value: DMSO

value: Alive cells stained with Hashtag 1 and Hashtag 2 and HSC pool (CD45RA-CD90+GFP+) stained with Hashtag 3 and HSC pool (CD45RA-CD90+GFP-) stained with Hashtag 4


title: Organoid, Patient 348, Coculture [JHH348CoOrg]

value: pancreas

value: PDAC

value: epithelium

value: coculture


title: MIAPACA-2, control 96, dox, rep 2

value: 96 hours

value: NTC


title: HT,input

value: WT


title: MIAPACA-2, HBS1L 48, dox, rep 2

value: 48 hours


title: MIAPACA-2, HBS1L 48, dox, rep 3


title: ATAC-Seq siIEL_FlcnKO5

description: sgFlcn siIEL OT-1 cells

source_name: sgFlcn-transduced OT-1 cells purified from the siIEL from the same host on day 7.5 post-LM-OVA infection


title: MIAPACA-2, HBS1L 48, dox, rep 1


title: Tumor-infiltrating lymphocytes,HTO

value: Flank bearing tumor tissue

value: lymphocytes


title: ATAC-Seq siIEL_Control5

description: sgNTC siIEL OT-1 cells

source_name: sgNTC-transduced OT-1 cells purified from the siIEL from the same host on day 7.5 post-LM-OVA infection


title: HT,Mcrs1ox,rep2

data_processing:
Raw fastq files were filtered and trimmed with fastp (v 0.22.0)
The filtered reads were aligned to GRCm38 reference genome with HISAT2 (v 2.2.1)
Raw counts were determined with featureCounts (v2.0.1).
The raw counts matrix was imported to R (v 4.2.1) . Data normalization and differential gene expression analysis were performed with the limma package.
Assembly: GRCm38
Supplementary files format and content: Table-delimited text files containing the raw counts of each sample
Supplementary files format and content: Xls files containing the differencially expression genes

growth_protocol: HT cells were cultured in RPMI-1640 media supplemented with 10% Fetal bovine serum and 1x Penicillin-Streptomycin.

extract_protocol:
The RNAsimple total RNA kit was used to extact total RNA according to the manufacturer’s instructions.
Total RNA was subjected to polyA selection and library preparation with a TruSeq Stranded mRNA LT Kit.


title: HS_p38i__FBC

description:
10x Genomics  feature barcode (FBC)
GEX_4_barcodes.tsv.gz
GEX_4_features.tsv.gz
GEX_4_matrix.mtx.gz

value: Cord blood

value: HSPC

value: Alive cells stained with Hashtag 1 and Hashtag 2 and HSC pool (CD45RA-CD90+) stained with Hashtag 3


title: ATAC-Seq siIEL_FlcnKO4

description: sgFlcn siIEL OT-1 cells

value: sgFlcn-transduced OT-1 cells purified from the siIEL from the same host on day 7.5 post-LM-OVA infection


title: HS_DMSO__FBC

description:
10x Genomics  feature barcode (FBC)
GEX_3_barcodes.tsv.gz
GEX_3_features.tsv.gz
GEX_3_matrix.mtx.gz


title: MIAPACA-2, HBS1L 48, no dox, rep 3


title: HT,Mcrs1ox,rep1

value: HT

value: Pancreatic cancer cells


title: MIAPACA-2, control 96, no dox, rep 2


title: UT_p38i__FBC

description:
10x Genomics  feature barcode (FBC)
GEX_2_barcodes.tsv.gz
GEX_2_features.tsv.gz
GEX_2_matrix.mtx.gz


title: ATAC-Seq siIEL_Control4

description: sgNTC siIEL OT-1 cells

value: sgNTC-transduced OT-1 cells purified from the siIEL from the same host on day 7.5 post-LM-OVA infection


title: MIAPACA-2, HBS1L 48, no dox, rep 2


title: HT,Control,rep3

value: Vector Control


title: MIAPACA-2, control 96, no dox, rep 1


title: ATAC-Seq siIEL_FlcnKO3

description: sgFlcn siIEL OT-1 cells


title: UT_DMSO__FBC

description:
10x Genomics  feature barcode (FBC)
GEX_1_barcodes.tsv.gz
GEX_1_features.tsv.gz
GEX_1_matrix.mtx.gz

value: DMSO


title: HT,Control,rep2


title: MIAPACA-2, HBS1L 48, no dox, rep 1


title: GE_DMSO_rpl1__FBC

description:
10x Genomics  feature barcode (FBC)
GEX_5_barcodes.tsv.gz
GEX_5_features.tsv.gz
GEX_5_matrix.mtx.gz

value: AAV6 with PGK-GFP cassette

value: Alive cells stained with Hashtag 1 and Hashtag 2 and HSC pool (CD45RA-CD90+GFP+) stained with Hashtag 3 and HSC pool (CD45RA-CD90+GFP-) stained with Hashtag 4


title: ATAC-Seq siIEL_Control3

description: sgNTC siIEL OT-1 cells


title: MIAPACA-2, PELO 48, dox, rep 3

value: PELO


title: GE_p38i_rpl2__GEX

description:
10x Genomics 3′ gene expression (GEX)
GEX_8_barcodes.tsv.gz
GEX_8_features.tsv.gz
GEX_8_matrix.mtx.gz

value: Alive cells stained with Hashtag 1 and Hashtag 2 and HSC pool (CD45RA-CD90+GFP+) stained with Hashtag 3 and HSC pool (CD45RA-CD90+GFP-) stained with Hashtag 5


title: HT,Mcrs1ox,rep3


title: HT,Control,rep1


title: ATAC-Seq siIEL_FlcnKO2

description: sgFlcn siIEL OT-1 cells


title: GE_p38i_rpl1__GEX

description:
10x Genomics 3′ gene expression (GEX)
GEX_7_barcodes.tsv.gz
GEX_7_features.tsv.gz
GEX_7_matrix.mtx.gz


title: Mcrs1oxYy1KO,ATAC,rep2

description:
Mcrs1oxYy1KO.bowtie2_aln.sorted.bigwig
Mcrs1oxYy1KO_peaks.narrowPeak
ATAC_peak_counts.xlsx

data_processing:
Raw fastq files were filtered and trimmed with fastp (v 0.22.0)
The filtered reads were aligned to the GRCm38 reference genome with Bowtie2(2.5.0)
Peak calling was performed using MACS2 (2.2.7.1) with the following parameters: --nomodel -s 1 --shift 75 --extsize 150 -q 0.01  -f BAMPE.
For track visualization, bigwig files were generated using deeptools(v 3.5.1) subcommand bamCoverage and normalized using FPKM.
Reads fall in peak regions were counted with bedtools(v 2.30.0).
Assembly: GRCm38
Supplementary files format and content: bigwig files for track visulization
Supplementary files format and content: NarrowPeak file
Supplementary files format and content: Read counts fall in peak regions

growth_protocol: Tumor cells were cultured in RPMI-1640 supplemented with 10% Fetal bovine serum (Wisent) and 1x Penicillin-Streptomycin.

extract_protocol:
1x10^4 cells were used as for nucleus isolation and Tn5 tagmention. Tagmented DNA was amplified by PCR using the Illumina sequencing adapters as primers.
ATAC-Seq library were generated using Vazyme Hyperactive ATAC-Seq Library Prep Kit for Illumina following manufacturer's instruction.


title: MIAPACA-2, PELO 48, dox, rep 2


title: GE_DMSO_rpl2__GEX

description:
10x Genomics 3′ gene expression (GEX)
GEX_6_barcodes.tsv.gz
GEX_6_features.tsv.gz
GEX_6_matrix.mtx.gz


title: MIAPACA-2, PELO 48, dox, rep 1


title: ATAC-Seq siIEL_Control2

description: sgNTC siIEL OT-1 cells


title: ATAC-Seq siIEL_FlcnKO1

description: sgFlcn siIEL OT-1 cells


title: ATAC-Seq siIEL_Control1

description: sgNTC siIEL OT-1 cells


title: HS_p38i__GEX

description:
10x Genomics 3′ gene expression (GEX)
GEX_4_barcodes.tsv.gz
GEX_4_features.tsv.gz
GEX_4_matrix.mtx.gz

value: WT


title: MIAPACA-2, PELO 48, no dox, rep 3

value: 48 hours


title: Mcrs1,ATAC,rep2

description:
Mcrs1ox.bowtie2_aln.sorted.bigwig
Mcrs1ox_peaks.narrowPeak
ATAC_peak_counts.xlsx


title: MIAPACA-2, PELO 48, no dox, rep 2


title: GE_DMSO_rpl1__GEX

description:
10x Genomics 3′ gene expression (GEX)
GEX_5_barcodes.tsv.gz
GEX_5_features.tsv.gz
GEX_5_matrix.mtx.gz


title: Mcrs1,ATAC,rep1


title: RNA-Seq S8_sgFlcn_day7.5

data_processing:
The Cell Ranger 6.0 Single-Cell Software Suite (10x Genomics) was implemented to process the  raw sequencing data from the Illumina HiSeq run.
This pipeline performed de-multiplexing,  alignment (mm10), and barcode processing to generate gene-cell matrices used for downstream  analysis.
Assembly: mm10
Supplementary files format and content: cell ranger count matrix for single cells in each sample

source_name: sgFlcn-transduced OT-1 cells purified from the siIEL on day 7.5 post-LM-OVA infection

growth_protocol: sgNTC or sgFlcn-transduced OT-1 cells purified from the siIEL on days 4.5 and 7.5 post-LM-OVA infection

extract_protocol:
CD8+ OT1 T cells were  loaded onto  the Chromium Controller according to their respective cell counts to target 9000 single cell GEMs (gel beads in emulsion) per sample.
Libraries were prepared using the Chromium Single Cell 3’ v3 Library and Gel Bead Kit (10x Genomics)

value: sgRNA transduced OT-1 cells


title: HS_DMSO__GEX

description:
10x Genomics 3′ gene expression (GEX)
GEX_3_barcodes.tsv.gz
GEX_3_features.tsv.gz
GEX_3_matrix.mtx.gz


title: Mcrs1oxYy1KO,ATAC,rep1


title: MIAPACA-2, control 48, dox, rep 3

value: NTC


title: Control,ATAC,rep1

description:
Control.bowtie2_aln.sorted.bigwig
Control_peaks.narrowPeak
ATAC_peak_counts.xlsx

value: sgControl


title: RNA-Seq S7_sgNTC_day7.5

source_name: sgNTC-transduced OT-1 cells purified from the siIEL on day 7.5 post-LM-OVA infection


title: UT_DMSO__GEX

description:
10x Genomics 3′ gene expression (GEX)
GEX_1_barcodes.tsv.gz
GEX_1_features.tsv.gz
GEX_1_matrix.mtx.gz

value: Cord blood

value: HSPC


title: RNA-Seq S6_sgFlcn_day7.5


title: MIAPACA-2, control 48, dox, rep 2


title: RNA-Seq S5_sgNTC_day7.5


title: CLIPseq_DDX10_rep2

city: guangzhou

first: gongcheng

last: Hu

state: guangdong

address: 190 Kai Yuan Avenue, Science Park, Guangzhou, China

institute: Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences

data_processing:
Single end reads with UMI sequences were selected. Fastx_clipper tool from fastx toolkit was used to remove 3’ linker sequence with parameters “-a TGGAATTCTCGGGTGCCAAGG -l 20 -n”. Fastq2collapse.pl from CLIP Tool Kit (CTK) was used to remove PCR duplicates, then subjected to stripBarcode.pl to remove 5’UMI sequences with parameters “-format fastq -len 4”.
Reads were mapped to mouse (mm10) genome with bwa. Sam files were parsed with parseAlignment.pl, tag2collapse.pl, joinWrapper.py tools from CTK, and then handled with tag2peak.pl to get enriched peaks, which was annotated with ChIPseeker. BamCoverage from deeptools was used to generate normalized bw files with parameters “--normalizeUsing RPGC --effectiveGenomeSize 2494787188”. Reads were also mapped to pre-45S DNA sequence (downloaded from NCBI: X82564) with bwa. BamCoverage was used to generate normalized bw files with parameters “--normalizeUsing RPGC --effectiveGenomeSize 22118”.
Assembly: mm10
Supplementary files format and content: Files containing annotation results of peaks derived from DDX10 CLIP-seq data.

growth_protocol: mESCs were cultured on gelatin-coated plates in DMEM high-glucose media supplemented with 15% FBS, 1 × GlutaMAX, 1 × nonessential amino acids, 1 × sodium pyruvate, 0.1 mM 2-mercaptoethanol, 3 μM CHIR99021, 1 μM PD0325901 and 1,000 U/mL leukemia inhibitory factor (LIF).

extract_protocol:
The RNAs were subjected to NuPAGE Bis-Tris gel separation and transferred to nitrocellulose membrane, and the biotin-labeled RNA-protein complexes were identified by chemiluminescent nucleic acid detection module kit. Finally, the RNAs were purified through proteinase K treatment and overnight precipitation.
The RNAs were ligated with 5’ RNA adaptor with T4 RNA Ligase1. Then the RNAs were reverse transcribed by Superscript III with RT primer and amplified for 15-18 cycles with Phusion High-Fidelity DNA Polymerase. Library index sequences were introduced by PCR with index primers for 8-10 cycles.

treatment_protocol: Cells were crosslinked with 400 mJ/cm2 of 254 nm UV light. Nuclear protein extracts were prepared from Flag-tagged DDX10 mESCs, and then treated with RQ1 DNase at 37°C for 3 min. About 50 μL Flag-M2 magnetic agarose beads (Sigma) were used for each purification. FLAG magnetic agarose beads  were incubated with lysate at 4°C overnight. Then the beads were partially digested with MNase (NEB, 1:5 × 105 dilution) at 37°C for 10 min. The reaction was stopped by PNK-EGTA buffer (PNK buffer with 2 mM EGTA). Then the beads were washed once with high salt buffer, once with wash buffer and twice with PNK buffer. To dephosphorylate the RNA, the beads were treated with FastAP thermosensitive alkaline phosphatase (Thermo Fisher Scientific) at 37°C for 20 min. The pre-adenylated 3’ linker was ligated with T4 RNA Ligase 2, truncated KQ (NEB) overnight at 16°C. The non-ligated 3’ linker was washed away with PNK buffer. The RNAs were then phosphorylated by T4 PNK (NEB) at 37°C for 30 min.

value: stably expressed Flag-tagged DDX10

tag: clip antibody


title: MIAPACA-2, PELO 48, no dox, rep 1

tag: cell line


title: MIAPACA-2, control 48, dox, rep 1


title: CLIPseq_DDX10_rep1

tag: genotype


title: RNA-Seq S4_sgFlcn_day4.5

source_name: sgFlcn-transduced OT-1 cells purified from the siIEL on day 4.5 post-LM-OVA infection


title: fetal pancreas, Sample5, 18PCW, snRNAseq

city: Toronto

first: Adriana

last: Migliorini

state: Ontario

address: 101 College St

department: McEwen Stem Cell Institute

country: Canada

institute: University Health Network

description: 10x Genomics v3.1

data_processing:
Alignment and gene counting were performed using Cell Ranger v3.1 (10X Genomics).
Transcript INS-IGF2 was removed from the GTF reference to unmask the INS transcripts.
Assembly: hg38
Supplementary files format and content: Feature barcode matrix in MTX format

source_name: human fetal pancreas

extract_protocol:
Single nuclei RNA seq was carried out using the 10X Genomic Platform. Briefly, 30-50mg of snap-frozen pancreatic tissue was cut in 1-2 mm3 on dry ice and transferred to cold lysis buffer (0.32mM sucrose, 5mM CaCl2, 3mM Mg (Ac)2, 20mM Tris-HCl, 0,1% Triton X-100, 0.1mM EDTA, and RNase Inhibitor (40U/ml)). The tissue was chopped and homogenized with strokes using a Dounce homogenizer until intact nuclei were visible upon SYBR Green Nucleic acid staining. Isolated nuclei were stained with DAPI and Fluorescent Activated Cell Sorting was used to enrich for intact nuclei. Sorted nuclei were used as input into the 10X Genomics single-cell 3’ v3.1 assay and processed as outlined by the user guide.
cDNA libraries were prepared as outlined by the Single Cell 3’ Reagent Kits v3.1 user guide with modifications to the PCR cycles based on the calculated cDNA concentration. The molarity of each library was calculated based on library size as measured using a bioanalyzer and qPCR amplification data. Samples were pooled and normalized to 1.5nM. The library pool was denatured using 0.2N NaOH for 8 minutes at room temperature and neutralized with 400mM Tris-HCL . Library pools at a final concentration of 300pM were loaded to sequence on NovaSeq 6000 (Illumina).

value: human fetal pancreas

tag: developmental stage

value: 18 PCW


title: MIAPACA-2, control 48, no dox, rep 3

tag: time


title: fetal pancreas, Sample6, 18PCW, snRNAseq

description: 10x Genomics v3.1


title: RNA-Seq S3_sgNTC_day4.5

source_name: sgNTC-transduced OT-1 cells purified from the siIEL on day 4.5 post-LM-OVA infection

tag: strain


title: RNAseq_auxin_2day_washoff_2day_rep2

data_processing:
Adaptor sequences were removed using Trim Galore and reads shorter than 20 nucleotides were discarded. Reads were then mapped to mouse (mm10) genome with STAR-RSEM pipeline using ENSEMBL version 79 gene annotation as reference.
Differential gene expression was determined using DESeq2. Genes with log2(fold change) > 1.5 and q value < 0.05 were considered significantly different.
Assembly: mm10
Supplementary files format and content: Tab-delimited text files include normalized count values from DESeq2.

growth_protocol: mESCs were cultured on gelatin-coated plates in DMEM high-glucose media supplemented with 15% FBS, 1 × GlutaMAX, 1 × nonessential amino acids, 1 × sodium pyruvate, 0.1 mM 2-mercaptoethanol, 3 μM CHIR99021，1 μM PD0325901 and 1,000 U/mL leukemia inhibitory factor (LIF).

extract_protocol:
Total RNAs were extracted with RNA Purification Kit PLUS (EZBioscience). mRNA was purified and used for the construction of sequencing libraries.
RNA sequencing libraries were constructed by using a VAHTS Universal V6 RNA-seq Library Prep Kit for Illumina (NR604-01/02) according to the manufacturer’s instructions.

treatment_protocol: To induce DDX10 degradation, indole-3-acetic acid (IAA) was added to the medium at a final concentration of 500 μM.

value: embryonic stem cells

value: Ddx10-AID, Tir1

tag: treatment

value: auxin-induced degradation of DDX10 2 days followed by withdrawal for 2 days


title: fetal pancreas, Sample4, 16PCW, snRNAseq

description: 10x Genomics v3.1

value: 16 PCW


title: MIAPACA-2, control 48, no dox, rep 1


title: RNAseq_auxin_2day_rep2

value: auxin-induced degradation of DDX10 2 days


title: RNA-Seq S2_sgFlcn_day4.5


title: UT_p38i__GEX

description:
10x Genomics 3′ gene expression (GEX)
GEX_2_barcodes.tsv.gz
GEX_2_features.tsv.gz
GEX_2_matrix.mtx.gz

value: Alive cells stained with Hashtag 1 and Hashtag 2 and HSC pool (CD45RA-CD90+) stained with Hashtag 3


title: RNAseq_auxin_2day_washoff_2day_rep1


title: Caco-2/15-Z2flag Subconfluent cells Exp2

city: TOULOUSE

first: Didier

last: Trouche

address: Université Toulouse III Paul Sabatier - Bât 4R4, 118 route de Narbonne

country: France

institute: Centre for Integrative Biology (CBI) of Toulouse MCD

data_processing:
Stranded paired-end reads were mapped to human rDNA genome and unmapped reads were aligned on hg38 using STAR (v2.5.2b) and processed using samtools keeping only primary alignement. Raw reads count were obtained using HT-seq.
Assembly: hg38 human genome
Supplementary files format and content: counts

source_name: Intestinal epithelium

growth_protocol: Samples for RNA-sequencing experiments were prepared in duplicates from subconfluent (Sc) or 7 days post-confluent (Pc) populations of Caco-2/15 cells expressing flagged version of H2A.Z.1 or H2A.Z.2 isoform.

extract_protocol:
We subjected the four samples of each (Sc or Pc) condition to strand-specific RNA-Seq method, relying on UTP incorporation in the second cDNA strand, as we previously described (Lamaa et al., 2020). Briefly, for each sample, 5–10 mg of total RNA, was extracted and submitted to EMBL-GeneCore (Heidelberg, Germany) for paired-end sequencing performed by Illumina’s NextSeq 500 technology. Two replicates of each sample were sequenced. The quality of each raw sequencing file (fastq) was checked with FastQC.
Stranded rRNA minus RNA-seq

value: Intestinal epithelium

value: Caco-2/15 cells

value: colon cancer cells

value: heterozygous H2AFV-Flag

value: Subconfluence


title: fetal pancreas, Sample3, 16PCW, snRNAseq

description: 10x Genomics v3.1


title: RNA-Seq S1_sgNTC_day4.5

value: sgRNA transduced OT-1 cells


title: fetal pancreas, Sample2, 14PCW, snRNAseq

description: 10x Genomics v3.1

value: 14 PCW


title: RNAseq_auxin_2day_rep1


title: Caco-2/15-Z2flag Subconfluent cells Exp1


title: RNAseq_auxin_1day_rep2

value: auxin-induced degradation of DDX10 1 day


title: brain, NMN03 (MCAO rats+NMN treatment)

city: Changchun

first: Daoyu

last: Zhang

address: Xinzhu street 1977

institute: Jilin University

data_processing:
We used STAR (https://github.com/alexdobin/STAR/archive/2.7.3a.tar.gz) to compare transcriptome data and cuffLinks (http://cole-trapnell-lab.github.io/cufflinks/) for quantitative splicing.
Assembly: hg38
Supplementary files format and content: the processed data files include TPM value for each sample

organism: Rattus norvegicus

source_name: rat brain

extract_protocol:
Total RNA was extracted with a kit(Qiagen),Concentration ≥30ng/μl, total amount ≥0.8μg, RIN≥5.8
Smart-Seq2 method was used to amplify each sample according to the manufacturer’s instructions.

treatment_protocol: Rats were randomly divided into three groups. Sham group: rats received surgery without MCAO; MCAO group (IR group): rats were subjected to MCAO; MCAO+NMN group (IR+NMN group): rats were subjected to MCAO and injected with 100 mg/kg NMN (Selleck, USA) in saline.

value: rat brain

tag: group

value: rat after MCAO and treatment with NMN

value: NMN


title: brain, NMN02 (MCAO rats+NMN treatment)


title: fetal pancreas, Sample1, 14PCW, snRNAseq

description: 10x Genomics v3.1

value: human fetal pancreas


title: Caco-2/15-Z2flag Postconfluent cells Exp2

value: 7-days postconfluence


title: CD4_Perturb-CITE-seq_GEX_08

city: San Francisco

first: Alexander

last: Marson

address: 1650 Owens St

institute: UCSF Gladstone Institutes

data_processing:
Fastqs for each 10X well were concatenated across lanes and flow cells.
Alignment of perturb-seq data and count aggregation for the gene expression, CRISPR guide, and Antibody Derived Tag (ADT) libraries was performed with cellranger54 count (v7.1.0) using the default settings and –expect-cells=45000 –chemistry=SC5P-R2.
Gene expression fastqs were aligned to “refdata-gex-GRCh38- 2020-A” human transcriptome reference acquired from 10x Genomics. Guide sequences were aligned to a custom reference file using the pattern TAGCTCTTAAAC(BC) while ADTs were aligned to the TotalSeq-C-Human-Universal-Cocktail-399905-Antibody-reference-UMI-counting.csv provided by Biolegend, also including the hashtag oligo (HTO) sequences which were used to distinguish each cell type and stimulation condition.
Counts for each respective library were aggregated across wells with cellranger aggr using the default settings.
Assembly: GRCh38
Supplementary files format and content: barcodes provided from cellranger after aggregation sourced from filtered_feature_bc_matrix
Supplementary files format and content: features provided from cellranger after aggregation sourced from filtered_feature_bc_matrix
Supplementary files format and content: Matrix provided from cellranger after aggregation sourced from filtered_feature_bc_matrix
Supplementary files format and content: sgrna calls per cell output from cellranger
Library strategy: Perturb-CITE-seq

source_name: Lymphocyte

growth_protocol: CD4+ regulatory and effector T cells were isolated from fresh Peripheral Blood Leukopaks (STEMCELL Technologies, #70500) from healthy human donors, after institutional review board–approved informed written consent (STEMCELL Technologies). The contents of the Leukopaks were washed twice with a 1X volume of EasySep buffer (DPBS, 2% fetal Bovine Serum (FBS), 1mM pH 8.0 EDTA) using centrifugation. The washed cells were resuspended at 200E6 cells/mL in EasySep buffer and isolated with the EasySep™ Human CD4+CD127lowCD25+ Regulatory T Cell Isolation Kit (STEMCELL Technologies, #18063), according to the manufacturer’s protocol. Following isolation with the kit, Tregs were stained Alexa Fluor® 647 anti-human CD25 Antibody (Biolegend, #302618), PE anti-Human CD127 (Beckon Dickinson, #557938), and Pacific Blue™ anti-human CD4 Antibody (Biolegend, #344620) and isolated using FACS to ensure a pure population without contaminating effector cells. After sorting pure CD4+CD127lowCD25+ Regulatory T Cells, the cells were seeded at 1x106 cells/mL in XVIVO-15 (Lonza, #02-053Q) supplemented with 55 uM 2-mercaptoethanol, 4 mM N-acetyl L-cysteine, and 200 U/mL IL-2 (Amerisource Bergen, #10101641). Teffs were seeded at 1x106 cells/mL in RPMI-1640 supplemented with 10% FCS, 2 mM L-Glutamine (Fisher Scientific #25030081), 10 mM HEPES (Sigma, #H0887-100ML), 1X MEM Non-essential Amino Acids (Fisher, #11140050), 1 mM Sodium Pyruvate (Fisher Scientific #11360070), 100 U/mL Penicillin-Streptomycin (Sigma, #P4333-100ML), and 50 U/mL IL-2 (Amerisource Bergen, #10101641). Both cell subsets were then stimulated with ImmunoCult™ Human CD3/CD28/CD2 T Cell Activator (STEMCELL Technologies, #10990) at 25 uL/mL for Tregs and 6.25 uL/mL for Teff. Following activation and electroporation, cells were split 1:2 every 48 hours to maintain an approximate density of 1x106 cells/mL and supplemented with respective doses of IL-2.

extract_protocol:
On the 10th day after initial isolation, the samples were collected for 10X single cell sequencing. First, cells from each donor within the same stimulation and cell type condition were pooled at equal concentrations. Sorting was performed to isolate live GFP+ cells from each condition. Sorted cells were processed according to the Chromium Next GEM Single Cell 5' HT Reagent Kits v2 (Dual Index) with Feature Barcode technology for CRISPR Screening and Cell Surface Protein guide User Guide, CG000513. In brief, sorted cells were pelleted and washed once with Cell Staining Buffer (Biolegend, #420201). Next, the samples were blocked with Human TruStain FcX™ Fc Blocking reagent (Biolegend, #422302). Meanwhile, TotalSeq™-C Human Universal Cocktail V1.0 (Biolegend, #399905) was prepared using Cell Staining Buffer (Biolegend, #420201) and TotalSeq™-C0251 anti-human Hashtag Antibodies 1-4 (Biolegend, #394661) were added to aliquots of the cocktail. HTOs were added to label the following populations: Treg Resting-1, Treg Stimulated- 2, Teff Resting-3, Teff Stimulated- 4. After blocking, cells were stained with TotalSeq-C cocktail including one Hashtag per cell and stimulation condition. After staining, the cells were washed three times in Cell Staining Buffer. The samples were then resuspended in PBS with 1% BSA (Gibco) for final counting. The resulting samples were pooled across conditions and approximately 65,000 cells per well were loaded into 8 wells of a Chromium Next GEM Chip N Single Cell Kit (10X Genomics, #1000375) for GEM generation.
The samples were prepared for sequencing using the Chromium Next GEM Single Cell 5' HT Kit v2 (#1000374), 5' Feature Barcode Kit, (#1000256), and 5' CRISPR Kit (#1000451) according to the manufacturer’s protocol. GEM generation and library preparation was performed by the Gladstone Genomics Core. The resulting libraries were sequenced using a NovaSeqX Series 10B flowcell (Illumina, #20085595) at the UCSF CAT.

treatment_protocol: 24 hours after stimulation of isolated human Tregs and Teffs from 2 donors, the cells were transduced with Zim3-dCas9 lentivirus at 3% v/v. The following day, Perturb-seq guide library lentivirus was added at 0.75% v/v (MOI 0.3). 48 hours after transduction with Zim3-dCas9, 10 mg/ml blasticidin (Gibco, #A1113903) was added to each sample to select for dCas9+ cells. Blasticidin was replenished every 48 hours until the cells were processed for sequencing. 8 days after initial isolation and stimulation of cells, half of the Treg and Teff culture was restimulated with ImmunoCult™ Human CD3/CD28/CD2 T Cell Activator (STEMCELL Technologies, #10990).

value: Lymphocyte

value: CD4+ T cells

tag: hto

tag: kit

value: GEM Single Cell 5' HT Kit v2 (#1000374)

tag: library type

value: GEX

tag: lane


title: CD4_Perturb-CITE-seq_GEX_07

tag: tissue

tag: cell type

value: TotalSeq™-C0251 anti-human Hashtag Antibodies 1-4 (Biolegend, #394661)


title: RNAseq_auxin_1day_rep1

value: embryonic stem cells

value: Ddx10-AID, Tir1


title: RNAseq_auxin_12h_rep2

value: auxin-induced degradation of DDX10 12 h


title: CD4_Perturb-CITE-seq_GEX_06


title: MIAPACA-2, control 48, no dox, rep 2

data_processing:
Reads were aligned using Illumina DRAGEN software.
Counts were calculated using featureCounts from subread package.
Lowly expressed counts were removed with the edgeR package (minimum count = 10).
Counts were adjusted by TMM normalization and limma-voom transformation.
Assembly: GRCh38.p13
Supplementary files format and content: .csv file containing raw counts for each sample
Supplementary files format and content: .csv file containing TMM-normalized log2 cpm values for each sample

extract_protocol:
RNA was harvested using Qiagen RNeasy Midi or Mini kit.
Libraries were prepped using the NEB Ultra II RNA library prep kit.

value: 48 hours

tag: sgrna

value: NTC

tag: doxycycline


title: RNAseq_auxin_12h_rep1


title: Caco-2/15-Z1flag Subconfluent cells Exp2

value: Intestinal epithelium

value: Caco-2/15 cells

value: colon cancer cells

value: heterozygous H2AFZ-Flag

value: Subconfluence


title: brain, IR03 (MCAO rats)

value: rat brain

value: rat after MCAO

value: PBS


title: Caco-2/15-Z2flag Postconfluent cells Exp1

value: heterozygous H2AFV-Flag

value: 7-days postconfluence


title: Caco-2/15-Z1flag Subconfluent cells Exp1


title: RNAseq_auxin_6h_rep2

value: auxin-induced degradation of DDX10 6 h


title: brain, IR02 (MCAO rats)


title: Caco-2/15-Z1flag Postconfluent cells Exp2


title: CD4_Perturb-CITE-seq_GEX_04

value: Lymphocyte

value: CD4+ T cells

value: TotalSeq™-C0251 anti-human Hashtag Antibodies 1-4 (Biolegend, #394661)

value: GEM Single Cell 5' HT Kit v2 (#1000374)

value: GEX


title: brain, IR01 (MCAO rats)


title: RNAseq_auxin_6h_rep1

value: embryonic stem cells

value: Ddx10-AID, Tir1


title: brain, Sham03

value: control


title: CD4_Perturb-CITE-seq_GEX_03


title: RNAseq_auxin_4h_rep2

value: auxin-induced degradation of DDX10 4 h


title: Caco-2/15-Z1flag Postconfluent cells Exp1

value: Intestinal epithelium

value: Caco-2/15 cells

value: colon cancer cells

value: heterozygous H2AFZ-Flag


title: CD4_Perturb-CITE-seq_GEX_02


title: brain, NMN01 (MCAO rats+NMN treatment)

value: rat brain

value: rat after MCAO and treatment with NMN

value: NMN


title: brain, Sham02

value: PBS


title: Caco-2/15-Z1flag Subconfluent cells Control Input

data_processing:
ChIP-seq reads were aligned to hg38 human genome using BWA and processed (sorting, PCR duplicates, minimum quality of 25 and indexing).
BigWig were obtained using Deeptools with exactScaling parameters and CPM normalization
Peaks were called uing MACS with the significance cut-off q-value <=0.001
Assembly: hg38 human genome
Supplementary files format and content: narrowPeak,bigWig

growth_protocol: Samples were prepared in duplicates from subconfluent (Sc) or 7 days post-confluent (Pc) populations of Caco-2/15 cells expressing flagged version of H2A.Z.1 or H2A.Z.2 isoform.

extract_protocol: For ChIP-seq experiments, Caco-2/15 cells expressing Flag-H2AZ.1 and Flag-H2AZ.2, 100 mg of chromatin supplemented with 10 ng of Spike-in chromatin (Active Motif) were used per ChIP experiment. For each reaction, 4 µg of Flag M2 antibody (Sigma) and 1 µg of Spike-in antibody (Active Motif), were used. About 10 ng of immunoprecipated DNA (quantified with quantiFluor dsDNA system, Promega) were obtained at each time, and samples were submitted to EMBL-GeneCore Heidelberg for sequencing, that was performed by Illumina’s NextSeq 500 technology (single-end, 85 bp reads). The quality of each raw sequencing file was verified with FastQC.

value: Subconfluence

tag: chip antibody


title: CD4_Perturb-CITE-seq_GEX_01

value: Lymphocyte

value: CD4+ T cells


title: RNAseq_auxin_2h_rep2

value: auxin-induced degradation of DDX10 2 h


title: RNAseq_auxin_4h_rep1


title: CD4_Perturb-CITE-seq_GEX_05

value: TotalSeq™-C0251 anti-human Hashtag Antibodies 1-4 (Biolegend, #394661)

value: GEM Single Cell 5' HT Kit v2 (#1000374)

value: GEX


title: Caco-2/15-Z2flag Subconfluent cells IP H2A.Z Exp2

value: heterozygous H2AFV-Flag

value: anti-H2A.Z (Active Motif #39113)


title: CD4_Perturb-CITE-seq_CSP_08

tag: adt

value: TotalSeq™-C Human Universal Cocktail V1.0 (Biolegend, #399905)

value: 5' Feature Barcode Kit, (#1000256)

value: CSP


title: Caco-2/15-Z2flag Subconfluent cells IP Flag Exp2

value: Intestinal epithelium

tag: cell line

value: Caco-2/15 cells

value: colon cancer cells

value: anti-Flag M2 (Sigma #F3165)