title: HTEN005, iPSC-MPC, Negative control, Day 7

city: Rochester

first: Hani

middle: A

last: Awad

address: 601 Elmwood Avenue

department: Biomedical Engineering

institute: University of Rochester

description: Library name: iMPC_NegCon_D7

data_processing:
Samples were aligned using CellRanger 8.0.0 using default parameters
Sequencing Platform - Illumina NovaSeqX Plus
Data Formatting - bcl-convert 4.1.7
Assembly: GRCh38
Supplementary files format and content: h5 filtered feature barcode matrix

source_name: Tenolysis tissue

growth_protocol: Primary fibroblasts were grown on tissue-culture treated plastic in MEM-alpha supplemented with 2% FBS, 1% pen-strep, and 55 microM 2-Mercaptoethanol. BM-MSCs and iMPCs were grown on tissue-culture treated plastic coated with ACF Cell Attachment Substrate (STEMCELL Technologies) in MesenCult-ACF Plus Media (STEMCELL Technologies). Cells were grown to 95% confluency prior to 21-day, 7-day, or tenogenic differentiation culture. At the end of culture, all cells were passaged with ACF Cell Dissociation Kit (STEMCELL Technologies) and resuspended as single cells prior to sequencing.

extract_protocol:
Extraction is included within the 10X genomics scRNA protocol
Cell suspensions were processed to generate single-cell RNA-Seq libraries using Chromium Next GEM Single Cell 3′ GEM, Library and Gel Bead Kit v3.1 (10x Genomics), per the manufacturer’s recommendations, as summarized below. Samples were loaded on a Chromium Single-Cell Instrument (10x Genomics, Pleasanton, CA, USA) to generate single-cell GEMs (Gel Bead-in-Emulsions). GEM reverse transcription (GEM-RT) was performed to produce a barcoded, full-length cDNA from poly-adenylated mRNA. After incubation, GEMs were broken, the pooled GEM-RT reaction mixtures were recovered, and cDNA was purified with silane magnetic beads (DynaBeads MyOne Silane Beads, ThermoFisher Scientific). The purified cDNA was further amplified by PCR to generate sufficient material for library construction.  Enzymatic fragmentation and size selection was used to optimize the cDNA amplicon size and indexed sequencing libraries were constructed by end repair, A-tailing, adaptor ligation, and PCR. Final libraries contain the P5 and P7 priming sites used in Illumina bridge amplification.

treatment_protocol: Tenogenic differentiation was performed using materials from STEMCELL Technologies. On day 0, cells were treated with a differentiation media A. This was changed to a differentiation media B on day 3. Media was refreshed every 2-3 days for the remaining culture period until day 21.

value: Tenolysis tissue

value: Fibroblast

value: Negative control 7 day culture


title: IFN15_Baseline_cd34pos_sn10x

status: Public on Jul 07 2026

type: SRA

city: New York

first: Anna

middle: S.

last: Nam

state: New York

address: Weiss Research Building, Floor 11

department: Pathology and Laboratory Medicine

country: USA

institute: Weill Cornell Medicine

description:
Library name: IFN15_Baseline_cd34pos_sn10x
10x genomics

library_source: genomic single cell

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

molecule: genomic DNA

organism: Homo sapiens

source_name: Bone marrow

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

tag: tissue

value: Bone marrow

tag: cell type

tag: disease state

value: Essential thrombocythemia

tag: treatment

value: Control


title: NSKO3

city: Montreal

first: Ana Maria

last: Hincapie

address: 1160 Pine avenue West

country: Canada

institute: McGill University

description: Library name: A007

library_source: transcriptomic

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

molecule: total RNA

organism: Mus musculus

source_name: Isolated B lymphocytes

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

value: Isolated B lymphocytes

tag: genotype

value: Ptpn2fl/fl mb1 Cre+

value: No treatment


title: HTEN005, Tenolysis fibroblast, Negative control, Day 7

state: NY

description: Library name: HTEN_NegCon_D7

library_source: transcriptomic single cell

molecule: polyA RNA

source_name: Mesenchymal progenitor cell differentiated from iPSC


title: NSKO2

description: Library name: A006


title: stomach male low-dose exposure rep4

city: Durham

first: Weichun

last: Huang

state: NC

address: 109 TW Alexander Dr

department: OCSPP

institute: US EPA

description: Library name: S_12

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

organism: Rattus norvegicus

source_name: stomach

growth_protocol: Rats were fed a standard rodent chow

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

value: stomach

tag: strain

value: Sprague-Dawley

tag: developmental stage

value: adult

value: WT

value: Male-Low Dose/5mg/kg

tag: batch

value: Batch 3


title: Bone marrow, MSC, Negative control, Day 7

description: Library name: BM-MSC_NegCon_D7

value: Mesenchymal stromal cell


title: stomach male low-dose exposure rep3

description: Library name: S_11

value: Batch 2


title: HTEN005, Tenolysis fibroblast, Negative control, Day 21

description: Library name: HTEN_NegCon_D21

value: Tenolysis tissue

value: Negative control 21 day culture


title: stomach male low-dose exposure rep2

description: Library name: S_10


title: HTEN005, Tenolysis fibroblast, 21-Day Tenogenic differentiation

description: Library name: HTEN_TenoDiff

value: Fibroblast

value: Tenogenic differentiation


title: stomach male low-dose exposure rep1

description: Library name: S_09


title: NSWT3

description: Library name: A003

value: Ptpn2wt/wt mb1 Cre+


title: HTEN005, iPSC-MPC, Negative control, Day 21

description: Library name: iMPC_NegCon_D21

value: Mesenchymal progenitor cell differentiated from iPSC


title: NSWT2

description: Library name: A002

value: Isolated B lymphocytes


title: stomach male control rep8

description: Library name: S_08

value: Male-Vehicle Control


title: RNA-seq_Differentiated KC,p63 siRNA,rep1

city: Stanford

first: Namyoung

last: Jung

state: CA

address: 269 Campus Dr. CCSR Room 2150

department: Medicine

institute: Stanford University

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

source_name: Primary keratinocyte

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

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

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

tag: cell line

value: Primary keratinocyte

value: Normal keratinocyte

value: p63 knockdown

value: Keratinocyte differentiation day3


title: HTEN005, iPSC-MPC, 21-Day Tenogenic differentiation

description: Library name: iMPC_TenoDiff


title: NSWT1

description: Library name: A001

value: No treatment


title: RNA-seq_Differentiated KC,control siRNA,rep2

value: WT


title: PDA, 30d, biol rep3

city: Changsha

first: Jiangshan

last: Ma

state: Hunan

address: No. 498 Shaoshan South Road, Tianxin District

country: China

institute: Central South University of Forestry and Technology

description: Library name: PDA30d-3

data_processing:
Raw sequencing reads were generated on the Illumina NovaSeq X Plus platform as 150 bp paired-end reads. Raw FASTQ files were processed using fastp to remove adapters and low-quality reads. Clean reads were aligned to the reference genome of Peniophora incarnata T-7 using HISAT2. Gene-level read counts were obtained using HTSeq-count, and normalized expression levels were calculated as FPKM values. Differential expression analysis was performed using DESeq2. GO, KEGG pathway, Reactome, and WikiPathways enrichment analyses of differentially expressed genes were performed based on the hypergeometric distribution using R.
Assembly: Peniophora incarnata T-7 genome assembly (GCA_040802745.1)
Supplementary files format and content: Gene-level normalized expression values were provided in a single Excel file (FPKM_anno.xlsx). The file contains FPKM values for all 12 RNA-seq samples, with rows representing genes and columns representing individual samples. Additional annotation columns include gene ID, gene symbol, gene description, GO annotation, and pathway annotation. FPKM values were calculated based on mapped reads to the reference genome.

organism: Peniophora incarnata

source_name: mycelium

growth_protocol: Peniophora incarnata T-7 was cultured at 28°C under constant temperature and humidity conditions (relative humidity 75%) for 30 days. For lignocellulose induction experiments, three types of lignocellulosic substrates (poplar wood, Chinese fir wood, and rice straw) were prepared as 60-mesh (≈250 μm) sieved biomass powders. All substrates were used as solid culture media, and fungal cultures were incubated in the dark under static conditions to allow colonization and degradation.

extract_protocol:
Total RNA was extracted from fungal mycelium using TRIzol reagent following the manufacturer’s instructions. RNA quality and concentration were assessed using a NanoDrop 2000 spectrophotometer, and RNA integrity was evaluated using an Agilent 2100 Bioanalyzer.
mRNA libraries were constructed using VAHTS Stranded mRNA-seq Library Prep Kit for Illumina. Poly(A)+ mRNA was enriched using oligo(dT) magnetic beads, followed by fragmentation, first- and second-strand cDNA synthesis, end repair, adapter ligation, size selection, and PCR amplification according to the manufacturer’s protocol.

treatment_protocol: Fungal cultures were grown on poplar wood (PW), Chinese fir wood (FW), rice straw (RS), and PDA medium as control (PDA) for 30 days. No additional chemical or environmental treatments were applied during incubation.

value: mycelium

value: fungal mycelium

value: wild type strain T-7

value: PDA medium for 30 days


title: Bone marrow, MSC, Negative control, Day 21

description: Library name: BM-MSC_NegCon_D21

value: Bone marrow

value: Mesenchymal stromal cell

value: Negative control 21 day culture


title: RNA-seq_Differentiated KC,control siRNA,rep1


title: stomach male control rep5

description: Library name: S_05

value: stomach

value: Sprague-Dawley

value: adult

value: Batch 1


title: RNA-seq_Undifferentiated KC,p63 siRNA,rep2

value: Primary keratinocyte

value: Keratinocyte differentiation day0


title: OralKC,Control,rep2

description: Library name: OralKC_rep2

value: Normal keratinocyte


title: PDA, 30d, biol rep2

description: Library name: PDA30d-2


title: PDA, 30d, biol rep1

description: Library name: PDA30d-1


title: RNA-seq_Undifferentiated KC,p63 siRNA,rep1

value: p63 knockdown


title: OralKC,Control,rep1

description: Library name: OralKC_rep1


title: RS, 30d, biol rep3

description: Library name: RS30d-3

value: rice straw for 30 days


title: RNA-seq_Undifferentiated KC,control siRNA,rep2

value: WT


title: OralKC,control,rep2

description: Library name: OralKC_rep2

library_source: genomic

data_processing:
Paired-end reads were aligned to the hg19 using HiC-Pro with default parameters.
To identify HiChIP peaks , hichipper was applied to the filtered reads using (EACH,ALL) setting. The HiC-Pro valid interactions and hichipper identified peaks were used to identify statistically significant loops using FitHiChIP with default parameters except for the following parameters: MappSize = 500, BINSIZE = 10000, QVALUE = 0.01, UseP2PBackground = 0.
Union of the significant loop list was generated and used for downstream differential loop analysis. The raw counts of all samples from FitHiChIP pipeline were processed with DESeq2 to identify differential loop between control siRNA treated and PAN-p63 siRNA treated samples.
Assembly: hg19
Supplementary files format and content: tab-delimited text file (output from FitHiChIP)
Library strategy: HiChIP

source_name: Primary oral keratinocyte

extract_protocol:
The HiChIP protocol was performed  as previously described (Mumbach et al., 2016) using  H3K27ac antibody  (Abcam, ab4729) with the following modifications. Samples were sheared using a Covaris E220 using the following parameters: Fill Level = 10, Duty Cycle = 5, PIP = 140, Cycles/Burst = 200, Time = 4 minutes and then clarified by centrifugation for 15 minutes at 16100 rcf at 4° C. We used 7.5 ug of antibody to H3K27ac and captured the chromatin-antibody complex with 60 uL Protein A beads (Thermo Fisher). Qubit quantification following ChIP ranged from 125-150 ng. The amount of Tn5 used and number of PCR cycles performed were based on the post-ChIP Qubit amounts.
HiChIP samples were size selected by PAGE purification (200-800 bp) .

value: Primary oral keratinocyte

tag: chip antibody


title: stomach male control rep2

description: Library name: S_02

value: Male-Vehicle Control

value: Batch 2


title: stomach male control rep7

description: Library name: S_07


title: A10-AD

city: Bethesda

first: Aaron

middle: M

last: Cypess

state: Maryland

address: 10 Center Drive, 5N318A

department: NIDDK - DEOB

institute: National Institutes of Health

description: Library name: sample_37

data_processing:
Data quality check was performed using FastQC (v0.11.8). The adapter trimming was performed using fastq-mcf program (v1.05) and cutadapt (v2.5). Contamination removal was performed using Bowtie2 (v2.5.1). Alignment was performed using STAR (v2.7.3a). The raw read counts were estimated using HTSeq (v0.11.2). Read count data were normalized using DESeq2. Additionally the aligned reads were used for estimating expression of the genes using cufflinks (v2.2.1). The expression values are also reported in FPKM (Fragments per kilobase per million) units for each gene. Additionally, RNA-Seq quality control was performed using RNA-SeQC (v1.1.8), RSeQC (v3.0.1) and MultiQC (v1.7).
Assembly: GRCh38/hg38
Supplementary files format and content: Excel spreadsheet containing DESeq2-normalized count
Supplementary files format and content: Salmon-based raw counts as tab-separated value file

source_name: periadrenal

extract_protocol:
Total RNA was extracted using RNeasy Kit (Qiagen, Germantown, MD) following the manufacturer’s protocol
Takara SMARTer Stranded Total RNA-Seq Kit v2 – Pico Input Mammalian

value: periadrenal


title: CHI191 Active Het

city: Memphis

first: Stanislav

last: Zakharenko

state: Tennessee

address: 262 Danny Thomas Pl, M2426

department: Developmental Neurobiology

institute: St. Jude Children's Research Hospital

description: Library name: CHI191

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

source_name: dorsal striatum

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

value: dorsal striatum

tag: group

value: Active

value: cholinergic interneuron

value: Het

tag: Sex

value: male

tag: date ephys

tag: dob

tag: age days

tag: electrophysiologist

tag: mouse id

tag: rt lot

tag: date lp

tag: lp lot

tag: index lot

tag: date seq

tag: seq srm_sample_id


title: RNA-seq_Undifferentiated KC,control siRNA,rep1

tag: cell type

tag: genotype

tag: treatment


title: RS, 30d, biol rep2

description: Library name: RS30d-2

tag: tissue

value: mycelium

value: fungal mycelium

value: wild type strain T-7


title: OralKC,control,rep1

description: Library name: OralKC_rep1

tag: cell line

tag: chip antibody


title: stomach male control rep1

description: Library name: S_01

value: stomach

tag: strain

value: Sprague-Dawley

tag: developmental stage

value: adult

tag: batch

value: Batch 1


title: RS, 30d, biol rep1

description: Library name: RS30d-1

value: rice straw for 30 days


title: CAL27,FOXK1 sgRNA,rep2

description: Library name: CAL27_FOXK1_KO_rep2

value: Squamous cell carcinoma

value: FOXK1 knockout


title: FW, 30d, biol rep3

description: Library name: FW30d-3

value: Chinese fir wood for 30 days


title: CAL27,FOXK1 sgRNA,rep1

description: Library name: CAL27_FOXK1_KO_rep1


title: duodenum female high-dose exposure rep7

description: Library name: D_47

source_name: duodenum

value: duodenum

value: WT

value: Female-High Dose/50mg/kg

value: Batch 2


title: FLASH 1x10Gy_ID567_Cortex_Other_002

city: Genève

first: Marie-Catherine

last: Vozenin

address: Avenue de la Roseraie 53

department: Radio-Oncologie

country: Switzerland

institute: HUG

description:
Library name: DSP-1001660035574-F-C07
FFPE fixed samples GeoMx Nanostring

data_processing:
DSP data were analyzed in R 4.4.0 (2024) according to Nanostring recommendation, using their in-house tools, with the exception of normalization which was performed using the trimmed mean of M values (TMM) algorithm of edgeR. Other packages were used for data manipulation and plotting as well as gene set enrichment analysis.
First, quality control (QC) was performed for the segments using a set of thresholds on chosen descriptive statistics of the segment. Chosen parameters were used as recommended by Nanostring. Then a Grubb test was performed for gene QC, also using parameters recommended by Nanostring. Next, negative probes were used to evaluate the background of quantification and define limit of quantification thresholds for filtering. Segments that contain less than 5% of the genes above the gene detection threshold and genes that are found in less than 5% of the segments were removed from the analysis. After filtering, normalization was performed using the TMM method of the edgeR package. Robust gene detection rate within segments and cell type specificity were confirmed, enabling downstream analysis.
Data was split per region (CA1, CA3, DG, SO and Cortex) and differential gene expression was performed using a linear mixed model (LMM) with the irradiation modality set as the test variable and the slide number as the random intercept.
All scripts used for the analysis and plotting can be found at https://gitlab.unige.ch/lirr/nanostring_flash_mouse_brain.
Assembly: MW0R30722
Supplementary files format and content: dcc
Library strategy: Spatial Transcriptomics

source_name: Brain

extract_protocol:
24h post-treatment completion, whole brains were collected and stored at 4°C in 4% PFA for 48 hours before Formalin-Fixed Paraffin-Embedded (FFPE) before processing
FFPE tissue section of 5-µm were processed according to the provider standard protocol for the GeoMx Digital Spatial Profiler (DSP).Neurons were identified using NeuN  (1:100, Millipore ABN78) revealed with a secondary goat anti-rabbit Alexa 594 (1:1000, Abcam ab150080). Vessels and astrocytes were also stained using an antibodies cocktail composed of CD31 Alexa 488 (Vessels, 1/100, R&D FAB3628G) and GFAP Alexa 647 (Astrocytes, 1/100, Novus NBP2-3318). The nuclear marker Syto83 (Invitrogen 511364) was also used. Whole slides were imaged at x20 magnification and morphologic markers were used to select regions of interest (ROI). Automatic segmentation of ROI based on CD31, NeuN and GFAP markers was performed to define area of illumination (AOIs). AOIs were exposed to 385 nm light (UV), releasing the indexing oligonucleotides to be sequenced. Sequencing libraries were generated by PCR from the photo-released indexing oligos. Pooled libraries were single-sequenced at 27 base pairs and with the single-index workflow  on an Illumina NovaSeq S4 instrument.

treatment_protocol: Irradiation was performed using a prototype 6 MeV electron beam, Oriatron type 6e (eRT6; PMB Alcen), LINAC, available at Lausanne University Hospital and described previously. Dosimetry has been extensively described and published to ensure reproducible and reliable biological studies. Animals were randomized in 5 groups and were sham or whole brain irradiated using a single dose of 10 Gy or 3 fractions of 10 Gy delivered 48 hours apart. A 17-mm graphite applicator was used and CONV was delivered at 0.09 Gy/s whereas FLASH was delivered in 1 pulse of 5.6x106Gy/s.

value: Brain

value: Other

value: Cortex

value: FLASH 1x10Gy


title: HiChIP_Differentiated KC,control siRNA,rep2

data_processing:
Paired-end reads were aligned to the hg19 using HiC-Pro with default parameters.
To identify HiChIP peaks , hichipper was applied to the filtered reads using (EACH,ALL) setting. The HiC-Pro valid interactions and hichipper identified peaks were used to identify statistically significant loops using FitHiChIP with default parameters except for the following parameters: MappSize = 500, BINSIZE = 10000, QVALUE = 0.01, UseP2PBackground = 0.
Union of the significant loop list was generated and used for downstream differential loop analysis. The raw counts of all samples from FitHiChIP pipeline were processed with DESeq2 to identify differential loop between control siRNA treated and PAN-p63 siRNA treated samples.
Assembly: hg19
Supplementary files format and content: tab-delimited text file (output from FitHiChIP)
Library strategy: HiChIP-seq

value: H3K27ac Abcam, ab472

value: Primary keratinocyte

value: Normal keratinocyte

value: Keratinocyte differentiation day3


title: duodenum female high-dose exposure rep6

description: Library name: D_46

value: Sprague-Dawley

value: adult

value: Batch 3


title: FW, 30d, biol rep1

description: Library name: FW30d-1

value: mycelium

value: fungal mycelium

value: wild type strain T-7


title: stomach male control rep6

description: Library name: S_06

value: stomach

value: WT

value: Male-Vehicle Control


title: CONV 1x10Gy_ID560_DG_Neurones_015

description:
Library name: DSP-1001660035574-F-B05
FFPE fixed samples GeoMx Nanostring

value: Brain

value: Neurones

value: DG

value: CONV 1x10Gy


title: CAL27,control sgRNA,rep1

description: Library name: CAL27_CTRL_rep1

value: Squamous cell carcinoma


title: CONV 1x10Gy_ID560_SO_Astrocytes_018

description:
Library name: DSP-1001660035574-F-C01
FFPE fixed samples GeoMx Nanostring

value: Astrocytes

value: SO


title: duodenum female high-dose exposure rep5

description: Library name: D_45

value: duodenum

value: Female-High Dose/50mg/kg

value: Batch 2


title: CONV 1x10Gy_ID560_CA3_Astrocytes_016

description:
Library name: DSP-1001660035574-F-B07
FFPE fixed samples GeoMx Nanostring


title: CONV 1x10Gy_ID560_CA1_Astrocytes_017

description:
Library name: DSP-1001660035574-F-B10
FFPE fixed samples GeoMx Nanostring


title: HiChIP_Undifferentiated KC,p63 siRNA,rep2

value: H3K27ac Abcam, ab472

value: Primary keratinocyte

value: Normal keratinocyte

value: p63 knockdown

value: Keratinocyte differentiation day0


title: CONV 1x10Gy_ID560_DG_Other_015

description:
Library name: DSP-1001660035574-F-B06
FFPE fixed samples GeoMx Nanostring

value: Other


title: duodenum female high-dose exposure rep4

description: Library name: D_44

value: Sprague-Dawley

value: adult

value: WT


title: HiChIP_Undifferentiated KC,p63 siRNA,rep1


title: iNKT cells, 41BB BCMA CAR, biol repl 2

city: London

first: Kanagaraju

last: Ponnusamy

address: Du Cane Road

department: Department of Immunology and Inflammation

country: United Kingdom

institute: Imperial College London

description:
Library name: SAMPLES 8
DEG_CD28 vs 41BB.xlsx
columns shows differentially expressed genes comparing CD28 BCMA CAR iNKT to 41BB BCMA CAR iNKT cells

data_processing:
RNA sequences were aligned using STAR
Differential analysis performed using DESeq2 R Bioconductor package
Assembly: GRCh38.101
Supplementary files format and content: csv file has all processed raw counts of RNA seq for all the smaples
Supplementary files format and content: excell file has output of DESeq2 for all the comparision

source_name: blood cells

growth_protocol: iNKT cells were activated by irradiated PBMCs and 100ng/1E6 cells anti-CD3 and anti-CD28 antibodies and expanded in 150U IL15 supplemented R10 media

extract_protocol:
total RNA isolated using Macherey-Nagel™ NucleoSpin™ RNA column and rDNase treatment on the column according to manufacturer’s instructions
mRNA library preparation (poly A enrichment)

treatment_protocol: lentiviral-mediated CAR expression

value: blood cells

value: primary cells

value: iNKT cells

value: 41BB BCMA CAR-transduced

value: transduced


title: PW, 30d, biol rep1

description: Library name: PW30d-1

value: mycelium

value: fungal mycelium

value: wild type strain T-7

value: poplar wood for 30 days


title: HiChIP_Undifferentiated KC,control siRNA,rep2

value: H3K27ac Abcam, ab472

value: Primary keratinocyte


title: iNKT cells, 41BB BCMA CAR, biol repl 1

description:
Library name: SAMPLES 7
DEG_CD28 vs 41BB.xlsx
columns shows differentially expressed genes comparing CD28 BCMA CAR iNKT to 41BB BCMA CAR iNKT cells


title: duodenum female high-dose exposure rep2

description: Library name: D_42

value: duodenum

value: Female-High Dose/50mg/kg

value: Batch 3


title: mm_rna_gcb_AIDcreBRWD1_DZ.2

city: Chicago

first: Mark

last: Maienschein-Cline

state: IL

address: 900 S Ashland Ave, Rm 1058, M/C 937

department: Research Resources Center

institute: University of Illinois at Chicago

data_processing:
Raw reads were aligned to reference genome mm9 in a splice-aware manner using STAR. Gene expression was quantified using featureCounts against UCSC genes, with Ensembl IG genes from mm10 converted to mm9 coordinates with UCSC liftOver.
Assembly: mm9
Supplementary files format and content: tab-delimited text file of raw counts per gene

source_name: Germinal Center DZd B cells B220+ GL7+ CD95+ CXCR4+ CD83-

extract_protocol:
Total RNA was isolated using a RNeasy kit (Qiagen).
Libraries were prepared by the University of Chicago Genomics Facility before sequencing.

treatment_protocol: Follicular B cell samples were isolated from mouse spleens. Follicular B cells were flow sorted as B220+ CD93+ CD21+ CD23+. Follicular B cells from Cd23-Cre Brwd1fl/fl mice were also sorted as tdTomato+. For Germinal Center B cell samples, mice were immunized intraperitoneally with 10^9 sheep red blood cells in PBS, then boosted with 10^9 sheep red blood cells 5 days later. On day 14, germinal center B cells were isolated from spleens. Germinal Center B cells were sorted as B220+ GL7+ CD95+ tdTomato+. LZ cells were defined as CXCR4- CD83+. DZp cells were defined as CXCR4+ CD83+. DZd B cells were defined as CXCR4+ CD83-.

value: Aicda-Cre Brwd1fl/fl

value: Mice were immunized intraperitoneally with 10^9 sheep red blood cells in PBS, then boosted with 10^9 SRBC 5 days later. On day 14, germinal center B cells were isolated from spleens.


title: PW, 30d, biol rep3

description: Library name: PW30d-3


title: HiChIP_Undifferentiated KC,control siRNA,rep1

value: Normal keratinocyte

value: WT

value: Keratinocyte differentiation day0


title: iNKT cells, 41BB BCMA CAR, biol repl 3

description:
Library name: SAMPLES 9
DEG_CD28 vs 41BB.xlsx
columns shows differentially expressed genes comparing CD28 BCMA CAR iNKT to 41BB BCMA CAR iNKT cells

value: blood cells

value: primary cells

value: iNKT cells


title: PW, 30d, biol rep2

description: Library name: PW30d-2

value: mycelium


title: iNKT cells, CD28 BCMA CAR, biol repl 3

description:
Library name: SAMPLES 6
DEG_NT vs 41BB.xlsx
columns shows differentially expressed genes comparing NT iNKT to 41BB BCMA CAR iNKT cells

value: CD28 BCMA CAR-transduced

value: transduced


title: mm_rna_gcb_AIDcreBRWD1_DZ.1

value: Germinal Center DZd B cells B220+ GL7+ CD95+ CXCR4+ CD83-


title: duodenum female high-dose exposure rep1

description: Library name: D_41

value: Sprague-Dawley

value: adult

value: Batch 1


title: duodenum female high-dose exposure rep3

description: Library name: D_43

value: duodenum

value: Female-High Dose/50mg/kg

value: Batch 2


title: iNKT cells, CD28 BCMA CAR, biol repl 2

description:
Library name: SAMPLES 5
DEG_NT vs 41BB.xlsx
columns shows differentially expressed genes comparing NT iNKT to 41BB BCMA CAR iNKT cells


title: duodenum female low-dose exposure rep8

description: Library name: D_40

value: Female-Low Dose/5mg/kg


title: HiChIP_SCC13,control siRNA,rep2

value: H3K27ac Abcam, ab472

value: Squamous cell carcinoma


title: HiChIP_Differentiated KC,p63 siRNA,rep2

value: Primary keratinocyte

value: Normal keratinocyte

value: p63 knockdown

value: Keratinocyte differentiation day3


title: mm_rna_gcb_AIDcreBRWD1_GZ.2

source_name: Germinal Center DZp B cells B220+ GL7+ CD95+ CXCR4+ CD83+

value: Aicda-Cre Brwd1fl/fl

value: Mice were immunized intraperitoneally with 10^9 sheep red blood cells in PBS, then boosted with 10^9 SRBC 5 days later. On day 14, germinal center B cells were isolated from spleens.


title: Differentiated KC,p63 siRNA,rep2

data_processing:
Sequencing data alignment, quality check, PCR duplicate removal, peak calling, blacklisted region removal, IDR peak filtration and signal track generation were performed through the ENCODE ATAC-seq pipeline (https://github.com/ENCODE-DCC/atac-seq-pipeline).
Assembly: hg19
Supplementary files format and content: tab-delimited text file includes peak coordinates and counts

extract_protocol:
55,000 viable cells were pelleted and resuspended in 50 uL of ATAC resuspension buffer (RSB) with 0.1% Igepal CA-630 (NP-40), 0.1% Tween-20, and 0.01% digitonin (Promega, G9441). After 3 minutes on ice, 1 mL of ATAC RSB with 0.1% Tween-20 was added, tubes were inverted, and nuclei pelleted by centrifugation at 500 RCF for 10 minutes at 4C. Supernatant was carefully removed and the nuclei pellet was resuspended in 50 uL of transposition mixture (25 uL TD buffer, 2.5 uL of TDE1 (Illumina, 20034197), 16.5 uL PBS, 0.5 uL 1% digitonin, 0.5 uL 10% Tween-20, 5 uL nuclease-free water). Transposition reactions were incubated at 37°C for 30 minutes in an Eppendorf ThermoMixer with agitation at 1000 RPM. Transposed DNA was purified using a Zymo DNA Clean and Concentrator-5 Kit (Zymo, D4014) and purified DNA was eluted in 20 ul elution buffer (10 mM Tris-HCl, pH 8).
ATAC-seq libraries were generated on two biological replicates for each group following Omni-ATAC protocol


title: duodenum female high-dose exposure rep8

description: Library name: D_48

value: WT


title: iNKT cells, CD28 BCMA CAR, biol repl 1

description:
Library name: SAMPLES 4
DEG_NT vs 41BB.xlsx
columns shows differentially expressed genes comparing NT iNKT to 41BB BCMA CAR iNKT cells

value: blood cells

value: primary cells

value: iNKT cells

value: CD28 BCMA CAR-transduced

value: transduced


title: HiChIP_Differentiated KC,p63 siRNA,rep1


title: duodenum female low-dose exposure rep7

description: Library name: D_39

value: duodenum

value: Sprague-Dawley

value: adult

value: Batch 3


title: mm_rna_gcb_AIDcreBRWD1_GZ.1

value: Germinal Center DZp B cells B220+ GL7+ CD95+ CXCR4+ CD83+


title: Differentiated KC,p63 siRNA,rep1

value: Primary keratinocyte


title: iNKT cells, NT, biol repl 3

description:
Library name: SAMPLES 3
DEG_NT vs CD28.xlsx
columns shows differentially expressed genes comparing NT iNKT to CD28 BCMA CAR iNKT cells

value: Non-transduced


title: duodenum female low-dose exposure rep6

description: Library name: D_38

value: Female-Low Dose/5mg/kg

value: Batch 2


title: mm_rna_gcb_AIDcreBRWD1_LZ.2

source_name: Germinal Center LZ B cells B220+ GL7+ CD95+ CXCR4- CD83+

value: Aicda-Cre Brwd1fl/fl


title: Differentiated KC,control siRNA,rep2

value: Normal keratinocyte

value: Keratinocyte differentiation day3


title: iNKT cells, NT, biol repl 2

description:
Library name: SAMPLES 2
DEG_NT vs CD28.xlsx
columns shows differentially expressed genes comparing NT iNKT to CD28 BCMA CAR iNKT cells

value: blood cells


title: duodenum female low-dose exposure rep5

description: Library name: D_37

value: WT

value: Batch 1


title: mm_rna_gcb_AIDcreBRWD1_LZ.1

value: Germinal Center LZ B cells B220+ GL7+ CD95+ CXCR4- CD83+

value: Mice were immunized intraperitoneally with 10^9 sheep red blood cells in PBS, then boosted with 10^9 SRBC 5 days later. On day 14, germinal center B cells were isolated from spleens.


title: Differentiated KC,control siRNA,rep1


title: iNKT cells, NT, biol repl 1

description:
Library name: SAMPLES 1
DEG_NT vs CD28.xlsx
columns shows differentially expressed genes comparing NT iNKT to CD28 BCMA CAR iNKT cells

value: primary cells

value: iNKT cells


title: duodenum female low-dose exposure rep4

description: Library name: D_36

value: duodenum

value: Sprague-Dawley

value: adult


title: Undifferentiated KC,p63 siRNA,rep2

value: Primary keratinocyte

value: p63 knockdown

value: Keratinocyte differentiation day0


title: FW, 30d, biol rep2

description: Library name: FW30d-2

value: mycelium

value: fungal mycelium

value: wild type strain T-7

value: Chinese fir wood for 30 days


title: Term

city: Shenzhen

first: Jun

last: Liu

state: Guangdong

address: No.13 Xinhu road

institute: Shenzhen Hospital of Southern Medical University

description:
Library name: sample189
10x Genomics

data_processing:
The raw data were preprocessed using Cell Ranger (version 7.0). The R toolkit Seurat (version 4.0.1) were used for downstream analysis
Assembly: hg19
Supplementary files format and content: Tab-separeted values files and matrix files

source_name: Peripheral blood

extract_protocol:
Single cells were suspended in calcium- and magnesium-free PBS containing 0.04% BSA after the peripheral blood mononuclear cells were isolated from the blood samples. Then, single cell suspension went through cell counting and viability assessment using Countess II FL Automated Cell Counter.
RNA libraries were prepared with chromium single cell 3 reagent kits v3 according to the manufacturer's protocol.

value: Peripheral blood

value: full-term infant


title: Relapsed-Rep5

city: Boston

first: Qingyu

last: Luo

state: MA

address: 450 Brookline Ave

institute: Dana-Farber Cancer Institute

description:
Library name: Sample 15
RRVA15

data_processing:
The raw FASTQ data were analyzed by the VIPER pipeline to generate gene expression data
Assembly: hg38
Supplementary files format and content: tab-delimited text file includes gene counts for each sample

source_name: bone marrow

extract_protocol:
Total RNA was extracted using NucleoSpin RNA Plus XS (740990.250; Takara Bio) according to the manufacturer’s protocol.
Takara SmartSeq v4

value: bone marrow

value: PDX

value: BPDCN

value: WT

value: venetoclax+azacitidine


title: duodenum female low-dose exposure rep3

description: Library name: D_35

value: duodenum

value: Female-Low Dose/5mg/kg

value: Batch 1


title: preterm

description:
Library name: sample141
10x Genomics

value: preterm infant


title: Undifferentiated KC,p63 siRNA,rep1

value: Primary keratinocyte

value: Normal keratinocyte

value: p63 knockdown


title: Relapsed-Rep4

description:
Library name: Sample 14
RRVA14


title: duodenum female low-dose exposure rep2

description: Library name: D_34

value: Sprague-Dawley

value: adult

value: Batch 2


title: Undifferentiated KC,control siRNA,rep2

value: Keratinocyte differentiation day0


title: HiChIP_SCC13,p63 siRNA,rep2

value: H3K27ac Abcam, ab472

value: Squamous cell carcinoma


title: CAL27,control sgRNA,rep2

description: Library name: CAL27_CTRL_rep2


title: Relapsed-Rep3

description:
Library name: Sample 13
RRVA13

value: bone marrow

value: PDX


title: duodenum female low-dose exposure rep1

description: Library name: D_33

value: WT


title: Undifferentiated KC,control siRNA,rep1


title: duodenum female control rep7

description: Library name: D_32

value: Female-Vehicle Control

value: Batch 3


title: SW480, P2RY2 KO, ATP treated, rep2

city: Heidelberg

first: Chong

last: Sun

state: Baden-Württemberg

address: Im Neuenheimer Feld 280

department: Immune Regulation in Cancer

country: Germany

institute: German Cancer Research Center

description:
Library name: Sample 17
Column name in all_cnt_FeatureCounts.tsv: SW480_KO_2

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

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

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

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

value: Colorectal cancer cells

value: P2RY2 knockout

value: 200 μM ATP


title: Relapsed-Rep2

description:
Library name: Sample 12
RRVA12

value: BPDCN

value: venetoclax+azacitidine


title: Rep2_PU1_MUT_Tx_CUT&RUN

description: CUT&RUN

data_processing:
10x data was processed using Cell Ranger 1 and v6.1.2 using default parameters. RNA reads were aligned to the human referece genome.
Amplicon reads was processed following steps: 1) Identification of reads with proper priming, 2) Identification of cell barcodes (CB) that match with the whitelisted CB, 3) Replacement of CB that are not perfectly matched with the whitelist CB, 4) Deduplication of reads, 5) Analyze reads with CB that are also observed in the 10x RNA-seq dataATAC-seq 6) Collapse networks of closely related UMIs within CBs to correct for sequencing error, 7) Matching of CB/UMI pairs found in 10x scRNA-seq data, discarding those matching non-CALR genes and those matching no gene below a supporting read threshold.
CUT&RUN data were down-sampled to have same number of reads across all conditions within the sample replicate to account for difference in sequencing depth. Among the down-sampled reads, low-quality reads were filtered out using Trimmomatic, resulting reads were aligned with hg38 genome with Bowtie2, and PCR duplicates were removed with SAMtools with a previously described workflow. Aligned reads were then used for peak calling with MACS3 with a q-value threshold of 0.01 and reads from IgG antibody as the control. SPI1 over-enrichment in the called peaks was confirmed with Simple Enrichment Analysis (SEA, v5.5.5). Overlapping peaks between replicates of the same condition were kept for further processing. The peaks were processed with multiBamSummary function from deepTools to obtain counts matrix per sample for downstream analysis.
Differential peak enrichment analysis was run between conditions (MUT vs WT and IFN⍺-treated vs control) using DESEQ2 for distal PU.1 peaks. Differential peaks in each condition were centered around SPI1 motif and motif enrichment was performed for regions +/- 250 bp from SPI1 motif using HOMER (v.4.1.1). Differential ranking between each motif two conditions was calculated.
Assembly: GRCh38
Supplementary files format and content: Processed 10X data is matrix table of gene/peak counts per cell for each sample
Supplementary files format and content: Output matrices processed from genotyping data have two columns: barcodes (BC) and Genotype

source_name: Bone marrow

extract_protocol:
GoT-IM Extraction Protocol: Cryopreserved bone marrow mononuclear cells approved by the Institutional Review Board of Weill Cornell Medicine were thawed and stained using standard procedures with the surface antibody CD34-PE (clone AC136, dilution 1:50, Miltenyi Biotec) and DAPI (Sigma-Aldrich), according to manufacturer’s protocol. To eliminate experimental batch effects, cells were labelled simultaneously with hashing antibodies with time-point-identifying barcodes using Hashtag Antibodies 1, 2 or 3 (TotalSeq-A, clone LNH-94, Biolegend) for GoT-IM. To link cell identities to expression of cell surface proteins, cells were also incubated with CITE-seq antibodies according to manufacturer protocol (TotalSeq-A, Biolegend). Cells were subsequently sorted for DAPI−, CD34+ cells using BD Influx at the Weill Cornell Medicine flow cytometry core. Genotyping of Transcriptomes (GoT) was performed by adapting the 10x Genomics platform. FACS-sorted CD34+ cells for each time-point from the same individual were pooled, targeting a total of 10,000-15,000 cells.
GoT-ATAC Extraction Protocol: Cryopreserved bone marrow mononuclear cells were thawed and stained using standard procedures with the surface antibody CD34-PE (clone AC136, dilution 1:50, MACS) and DAPI (Sigma-Aldrich), according to manufacturer’s protocol. Cells were subsequently sorted for DAPI−, CD34+ cells using BD Influx at the Weill Cornell Medicine flow cytometry core. Nuclei were isolated from DAPI-, CD34+ cells according to 10x Genomics Demonstrated Low Cell Input Nuclei Isolation protocol. Lysis buffer was prepared following manufacturer’s recommendations and then split into aliquots for each serial sample. Either TotalSeq-A Anti-Nuclei Pore Complex Proteins Hashtag 9 or 10 Antibody (1uL at 1:5 dilution; BioLegend) was added to each aliquot of lysis buffer. Low-input nuclei isolation was otherwise performed following manufacturer’s recommendations. Subsequently, nuclei from each time-point were counted and pooled together at approximately equal proportions. For IFN03, additional nuclei from the IFNa-treated sample were available to be run on a separate lane.
RFX3-OE Extraction Protocol: Cells from frozen umbilical cord blood mononuclear cells were thawed rapidly at 37°C, red blood cell lysis was performed using ACK Lysis Buffer, then centrifuged, and cell pellet was resuspended in cold MACS buffer. Cells were centrifuged at 300g for 5min at 4°C, and resuspended in cold MACs buffer at a concentration of 1x10^7 cells/mL. CD34+ cells were then isolated with the EasySep™ Human CD34 Positive Selection Kit II (StemCell Technologies #17856) according to manufacturer’s protocol. After isolation, cells were counted and plated in 96-well round-bottom plates (Falcon) in StemSpan™ SFEM II media (StemCell Technologies) with StemSpan™ CD34+ Expansion Supplement (StemCell Technologies) and 1% Pen/Strep. After 24 hours in culture, cells were spun down and resuspended in minimal media consisting of StemSpan StemSpan™ SFEM II media with StemSpan™ CD34+ Expansion Supplement, 1% Pen/Strep, 10μM prostaglandin E2 (StemCell Technologies #72192) and 100ng/μl poloxamer 407 (Millipore Sigma P2164030). Cells were split into 2 technical replicates each for untransduced condition, mCherry-control vector transduction, and RFX3-OE vector transduction. Lentivirus or PBS control was added at an MOI of 100, spinoculated at 300g, 32°C for 1 hour, and cells were incubated in same lentivirus-containing media for 24 hours at 37°C before resuspension in fresh media consisting of StemSpan™ SFEM II media with StemSpan™ CD34+ Expansion Supplement and 1% Pen/Strep. After 48 hours of incubation, cells were stained with CD34-APC antibody (1:50). Cells were then FACS isolated for the CD34+ population in the untransduced condition and the CD34+ mCherry+ populations in the RFX3-OE and mCherrycontrol conditions. Three independent transduction experiments were completed with new units of umbilical cord blood cells. Untransduced, mCherry-Control, and RFX3-OE CD34+ cells were stained with Cell Hashing antibodies (HTO TotalSeq-A, BioLegend) for 30 min at 4°C. Cells were then washed with FACs Buffer three times, pooled, and counted for loading. scRNA-sequencing was then performed using the 10x 3’ v3.1 platform according to manufacturer’s protocol. HTO demultiplexing and downstream analyses such as differential gene expression and gene set enrichment were performed as in GoT-IM for CD34+ bone marrow samples. scRNA-seq experiment was completed on one replicate of three independent transduction experiments.
CUT&RUN Extraction Protocol: UT7 cell lines expressing MPL (thrombopoietin receptor) and either the mutant CALR (type 1, L367Tfs*46) or wildtype CALR transgene (93) were seeded in 10cm plates in DMEM supplemented with 10% FBS (Thomas Scientific) and 5 ng/ml GM-CSF (Miltenyi-Biotec). Cells were treated with 0.1μg/ml Recombinant Human IFN-alpha2a (RC217-14) or PBS-1X control for 24hrs. After treatment, cells were washed 1x with PBS and collected for use with the CUTANA™ CUT&RUN assay (EpiCypher) according to the manufacturer’s protocol. 0.5μg of anti-human rabbit monoclonal PU.1 antibody (ab76543) and IgG antibody (ab172730) were used in the assay.
GoT-IM Library Construction Protocol: The standard 10x Genomics Chromium 3′ (v.3.1 chemistry) was implemented according to the manufacturer’s recommendations up to the cDNA amplification step (10X Genomics, Pleasanton, CA). After cDNA amplification and SPRI cleanup, 10x scRNA-seq and ADT/HTO libraries were generated as recommended. A portion of the cDNA was used for somatic genotyping. Briefly, to capture the somatic genotypes of cells, cDNA was amplified with a locus-specific amplification (10 PCR cycles), using the generic forward SI-PCR primer and a following locus-specific reverse primer for the CALR mutations. The amplified locus-specific cDNAs are then cleaned using SPRI purification to remove unincorporated primers. Finally, the targeted amplicon libraries are generated through a PCR performed with a P5 generic forward PCR primer together with an RPI-x primer. The targeted amplicon libraries were spiked into the remainder of the gene expression and immunophenotyping libraries to be sequenced together on a NovaSeq (Illumina, San Diego, CA). The cycle settings were as follows: 28 cycles for Read 1, 90 cycles for Read 2, 8 cycles for i7 sample index and 8 cycles for i5 sample index.
GoT-IM Library Construction Protocol: The standard 10x Genomics Chromium 3′ (v.3.1 chemistry) was implemented according to the manufacturer’s recommendations up to the cDNA amplification step (10X Genomics, Pleasanton, CA). After cDNA amplification and SPRI cleanup, 10x scRNA-seq and ADT/HTO libraries were generated as recommended. A portion of the cDNA was used for somatic genotyping. Briefly, to capture the somatic genotypes of cells, cDNA was amplified with a locus-specific amplification (10 PCR cycles), using the generic forward SI-PCR primer and a following locus-specific reverse primer for the CALR mutations. The amplified locus-specific cDNAs are then cleaned using SPRI purification to remove unincorporated primers. Finally, the targeted amplicon libraries are generated through a PCR performed with a P5 generic forward PCR primer together with an RPI-x primer. The targeted amplicon libraries were spiked into the remainder of the gene expression and immunophenotyping libraries to be sequenced together on a NovaSeq (Illumina, San Diego, CA). The cycle settings were as follows: 28 cycles for Read 1, 90 cycles for Read 2, 8 cycles for i7 sample index and 8 cycles for i5 sample index.
GoT-ATAC Library Construction Protocol: Single-nucleus gene expression (GEX) and chromatin accessibility libraries were constructed from the pooled nuclei according to the Chromium Next GEM Single Cell Multiome User Guide (10X Genomics). Genotyping libraries targeting the CALR mutant transcripts were constructed from the remaining amplified cDNA, similar to the original GoT method. For each PCR, 12.5uL Kapa HiFI HotStart Ready Mix was mixed with 0.75uL of 10uM forward primer, 0.75uL of 10uM reverse primer, 3uL cDNA and nuclease-free water for a total reaction volume of 25uL. In the first PCR, 3uL cDNA was re-amplified with Partial TSO and Partial Read 1 primers, using the following PCR condition: 98°C for 3min; 3 cycles of 98°C for 15sec, 67°C for 20sec and 72°C for 1min; 72°C for 1min. The re-amplified sample was purified and concentrated via 0.7X SPRI cleanup, eluting it into 10uL Buffer EB. To pre-enrich the CALR mutation locus, a gene-specific PCR was performed with 3uL of cleaned re-amplified cDNA and Partial Read 1 and gene-specific primers. The following PCR condition was used: 98°C for 3min; 11 cycles of 98°C for 20sec, 60°C for 20sec, and 72°C for 2min; 72°C for 2min. After 0.7X SPRI cleanup, cDNA was eluted into 10uL Buffer EB. CALR locus-specific amplification was then performed with 3uL of cleaned gene-specific amplified cDNA and SI-PCR and loci-specific Primers, using the PCR condition: 98°C for 3min; 11 cycles of 98°C for 20sec, 60°C for 20sec, and 72°C for 2min; 72°C for 2min. A 0.7X SPRI cleanup was performed, and cDNA was eluted into 11uL Buffer EB. Finally, to construct the targeted amplicon library, loci-amplified cDNA was mixed with P5 Generic and RPIx indexing primers and amplified with the PCR condition: 98°C for 3min; 5 cycles of 98°C for 15sec, 60°C for 20sec, and 72°C for 1min; 72°C for 1min. The constructed library was cleaned via 0.8X SPRI cleanup and eluted into 12uL Buffer EB. At the cDNA amplification stage of the Chromium Next GEM Single Cell Multiome protocol, supernatant from the 0.6X size selection was retained and was used to generate the hashing libraries as per HTO protocol25 with the following modification. For the hashing library construction step, the PCR reaction was prepared with 0.65uL of 10uM SI-PCR primer, 0.65uL of 10uM TruSeq DNA D7xx_s primer, 11.25uL cleaned supernatant and 12.5 uL Kapa HiFI HotStart Ready Mix (Roche, Basel, Switzerland). Hashing, gene expression and genotyping libraries were pooled and sequenced together on a NovaSeq (Illumina) with cycle settings: 28 cycles for Read 1, 90 cycles for Read 2, 10 cycles for i7 sample index and 10 cycles for i5 sample index. The ATAC library was sequenced separately on a NovaSeq, with cycle settings: 50 cycles for Read 1 and Read 2, 8 cycles for i7 sample index and 24 cycles for i5 sample index. Single-cell RNA-seq (10X) with targeted amplification of a mutation of interest.

value: Bone marrow

tag: disease state

value: Chronic Myelogenous Leukemia Cml

tag: library type

value: CUT&RUN


title: HiChIP_SCC13,control siRNA,rep1


title: Relapsed-Rep1

description:
Library name: Sample 11
RRVA11


title: duodenum female control rep6

description: Library name: D_31

value: duodenum

value: Sprague-Dawley

value: adult

value: Batch 2


title: HiChIP_CAL27,p63 siRNA,rep2

value: H3K27ac Abcam, ab472

value: Squamous cell carcinoma

value: p63 knockdown


title: Rep2_PU1_MUT_Untx_CUT&RUN

description: CUT&RUN

value: Control

value: IFNa


title: Residual-Rep5

description:
Library name: Sample 10
RRVA10

value: bone marrow

value: PDX

value: WT


title: duodenum female control rep5

description: Library name: D_30

value: Female-Vehicle Control


title: HiChIP_CAL27,p63 siRNA,rep1


title: Residual-Rep4

description:
Library name: Sample 9
RRVA9

value: BPDCN

value: venetoclax+azacitidine


title: Rep2_PU1_WT_Tx_CUT&RUN

description: CUT&RUN

value: Bone marrow

value: Chronic Myelogenous Leukemia Cml


title: duodenum female control rep4

description: Library name: D_29

value: duodenum

value: Batch 1


title: HiChIP_CAL27,control siRNA,rep2


title: Residual-Rep3

description:
Library name: Sample 8
RRVA8


title: duodenum female control rep3

description: Library name: D_28

value: Sprague-Dawley

value: adult


title: Rep2_PU1_WT_Untx_CUT&RUN

description: CUT&RUN

value: CUT&RUN


title: HiChIP_CAL27,control siRNA,rep1

value: H3K27ac Abcam, ab472

value: Squamous cell carcinoma


title: Rep1_PU1_MUT_Tx_CUT&RUN

description: CUT&RUN


title: HiChIP_Differentiated KC,control siRNA,rep1

value: Primary keratinocyte

value: Normal keratinocyte

value: Keratinocyte differentiation day3


title: Residual-Rep2

description:
Library name: Sample 7
RRVA7

value: bone marrow

value: PDX


title: SCC13,p63 ChIP,rep2

description:
SCC13_p63_ChIP_rep_mean.bw
SCC13_p63_ChIP.bed

data_processing:
The sequencing reads were aligned to the hg19 genome with Bowtie2 with default parameters.
Aligned reads were filtered for MAPQ>30 and PCR duplicates with SAMtools(1.8). Peak calling was performed with MACS2 with p value less than 0.1 cutoff.
These peaks were then subjected to Irreproducible Discovery Rate (IDR) analysis to identify reproducible peaks with IDR cutoff of 0.05.
The final peak set was determined by combining the IDR filtered peaks and the common peak between the two biological replicates.
Assembly: hg19
Supplementary files format and content: BED, BigWig

growth_protocol: CAL27 human squamous cell carcinoma cells were obtained from ATCC (CRL-2095) and cultured in DMEM F:12 (ThermoFisher) supplemented with 10% FBS. SCC-13 human squamous cell carcinoma cells were obtained from J.G.Rheinwald, Dana-Farber/Harvard Cancer Center as a generous gift and cultured in Keratinocyte-SFM (ThermoFisher) supplemented with 25 ug/ml BPE, 0.2ng/ml EGF (GIBCO, Thermofisher) and 0.3mM CaCl2 (Sigma-Aldrich).

extract_protocol:
Briefly, 20-60 million cells of SCCs were harvested and crosslinked with 1% formaldehyde (ThermoFisher) for 10min and quenched with final 0.125 M glycine. The cross-linked cells were lysed with cell lysis buffer (50mM Tris pH 7.5, 150mM NaCl, 5mM EDTA, 1% Triton X-100, 0.5% NP-40) containing protease inhibitor on ice for 10min. The extracted nuclei were pelleted by centrifugation and then resuspended in 1ml of nuclei lysis buffer (50mM Tris pH 7.5, 10mM EDTA, 1% SDS) containing protease inhibitor. The nuclei were sheared using Bioruptor (Diagenode) for 40 min. The shear chromatin was cleared by centrifugation. The supernatant was transferred and diluted with 2-fold volume of ChIP dilution buffer (16.7mM Tris pH 7.5, 167mM NaCl, 1.2mM EDTA, 1.1% of Triton X-100, 0.01% of SDS) containing protease inhibitor. The diluted chromatin was incubated with Dynabeads Protein A (Thermo Fisher) for 1hr at 4°C to pre-clear the chromatin. The pre-cleared chromatin was then incubated with 10ug of p63 antibody (Abcam) for overnight at 4°C. 60ul per 10million cells of Dynabeads were incubated with ChIPed samples for 3hrs at 4°C with rotation. The beads were washed with RIPA-low salt buffer(50mM Tris pH 8.0, 1mM EDTA, 150mM NaCl, 1% Triton X-100, 0.1% SDS, 0.1% sodium deoxycholate), RIPA-high salt buffer(50mM Tris-8.0, 1mM EDTA, 500mM NaCl, 1% Triton X-100, 0.1% SDS, 0.1% sodium deoxycholate), LiCl wash buffer (50mM Tris pH8.0,250mM LiCl, 0.5% NP-40, 0.5% sodium deoxycholate) and TE buffer(10mM Tris pH 8.0, 1mM EDTA) twice. The ChIPed DNA was eluted with100ul Elution buffer (10mM Tris pH8.0, 5mM EDTA, 300mM NaCl, 0.5% SDS) twice by incubation at 65 °C for 15 min twice. For reverse crosslinking and purification, 10ul of proteinase K (NEB) was added to the eluted ChIPed DNA and incubated for 1hr at 55°C and then at least 4hrs at 67°C with shaking. To remove RNA, 5ul of RNase A (Thermo Fisher) was added and incubated at 37°C for 30min.
The reverse crosslinked DNA was purified with Zymo ChIP DNA Clean & Concentrator (Zymo research) and library was generated using NEBnext ultra II kit(NEB) according to the manufacturer’s protocol.

value: WT


title: duodenum female control rep2

description: Library name: D_27

value: Female-Vehicle Control


title: Rep1_PU1_MUT_Untx_CUT&RUN

description: CUT&RUN

value: Control


title: Residual-Rep1

description:
Library name: Sample 6
RRVA6

value: BPDCN

value: venetoclax+azacitidine


title: duodenum female control rep1

description: Library name: D_25

value: duodenum

value: Batch 2


title: Rep1_PU1_WT_Tx_CUT&RUN

description: CUT&RUN

value: Bone marrow

value: Chronic Myelogenous Leukemia Cml

value: IFNa


title: SCC13,p63 ChIP,rep1


title: duodenum male high-dose exposure rep8

description: Library name: D_24

value: Sprague-Dawley

value: adult

value: Male-High Dose/50mg/kg

value: Batch 1


title: Primary-Rep5

description:
Library name: Sample 5
RRVA5

value: Vehicle


title: SCC13,none,rep2

value: Squamous cell carcinoma


title: Primary-Rep4

description:
Library name: Sample 4
RRVA4

value: bone marrow

value: PDX


title: duodenum male high-dose exposure rep7

description: Library name: D_23

value: WT


title: Rep1_PU1_WT_Untx_CUT&RUN

description: CUT&RUN

value: Control

value: CUT&RUN


title: RFX3_OE_cd34pos_10x

description: 10x genomics

library_source: other

data_processing:
10x data was processed using Cell Ranger 1 and v6.1.2 using default parameters. RNA reads were aligned to the human referece genome.
Amplicon reads was processed following steps: 1) Identification of reads with proper priming, 2) Identification of cell barcodes (CB) that match with the whitelisted CB, 3) Replacement of CB that are not perfectly matched with the whitelist CB, 4) Deduplication of reads, 5) Analyze reads with CB that are also observed in the 10x RNA-seq dataATAC-seq 6) Collapse networks of closely related UMIs within CBs to correct for sequencing error, 7) Matching of CB/UMI pairs found in 10x scRNA-seq data, discarding those matching non-CALR genes and those matching no gene below a supporting read threshold.
CUT&RUN data were down-sampled to have same number of reads across all conditions within the sample replicate to account for difference in sequencing depth. Among the down-sampled reads, low-quality reads were filtered out using Trimmomatic, resulting reads were aligned with hg38 genome with Bowtie2, and PCR duplicates were removed with SAMtools with a previously described workflow. Aligned reads were then used for peak calling with MACS3 with a q-value threshold of 0.01 and reads from IgG antibody as the control. SPI1 over-enrichment in the called peaks was confirmed with Simple Enrichment Analysis (SEA, v5.5.5). Overlapping peaks between replicates of the same condition were kept for further processing. The peaks were processed with multiBamSummary function from deepTools to obtain counts matrix per sample for downstream analysis.
Differential peak enrichment analysis was run between conditions (MUT vs WT and IFN⍺-treated vs control) using DESEQ2 for distal PU.1 peaks. Differential peaks in each condition were centered around SPI1 motif and motif enrichment was performed for regions +/- 250 bp from SPI1 motif using HOMER (v.4.1.1). Differential ranking between each motif two conditions was calculated.
Assembly: GRCh38
Supplementary files format and content: Processed 10X data is matrix table of gene/peak counts per cell for each sample
Supplementary files format and content: Output matrices processed from genotyping data have two columns: barcodes (BC) and Genotype
Library strategy: GEX+ADT+HTO

source_name: Umbilical cord

value: Umbilical cord

value: RFX3-OE, Control

value: GEX+ADT+HTO


title: SCC13,none,rep1


title: CAL27,p63 ChIP,rep2

description:
CAL27_p63_ChIP_rep_mean.bw
CAL27_p63_ChIP.bed


title: Primary-Rep3

description:
Library name: Sample 3
RRVA3

value: BPDCN


title: duodenum male high-dose exposure rep6

description: Library name: D_22

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

value: duodenum


title: HU02_cd34pos_10x

description: 10x genomics

value: Bone marrow

value: Essential thrombocythemia

value: HU


title: Primary-Rep2

description:
Library name: Sample 2
RRVA2


title: duodenum male high-dose exposure rep5

description: Library name: D_21

value: Sprague-Dawley

value: adult

value: Male-High Dose/50mg/kg

value: Batch 2


title: CAL27,p63 ChIP,rep1

value: Squamous cell carcinoma


title: HU01_cd34pos_10x

description: 10x genomics


title: Primary-Rep1

description:
Library name: Sample 1
RRVA1

value: bone marrow

value: PDX

value: Vehicle


title: stomach male high-dose exposure rep4

description: Library name: S_20

value: stomach

value: WT

value: Batch 1


title: duodenum male high-dose exposure rep4

description: Library name: D_20


title: IFN13_cd34pos_snGenotyping

description: Ironthrone-GoT

data_processing:
10x data was processed using Cell Ranger 1 and v6.1.2 using default parameters. RNA reads were aligned to the human referece genome.
Amplicon reads was processed following steps: 1) Identification of reads with proper priming, 2) Identification of cell barcodes (CB) that match with the whitelisted CB, 3) Replacement of CB that are not perfectly matched with the whitelist CB, 4) Deduplication of reads, 5) Analyze reads with CB that are also observed in the 10x RNA-seq dataATAC-seq 6) Collapse networks of closely related UMIs within CBs to correct for sequencing error, 7) Matching of CB/UMI pairs found in 10x scRNA-seq data, discarding those matching non-CALR genes and those matching no gene below a supporting read threshold.
CUT&RUN data were down-sampled to have same number of reads across all conditions within the sample replicate to account for difference in sequencing depth. Among the down-sampled reads, low-quality reads were filtered out using Trimmomatic, resulting reads were aligned with hg38 genome with Bowtie2, and PCR duplicates were removed with SAMtools with a previously described workflow. Aligned reads were then used for peak calling with MACS3 with a q-value threshold of 0.01 and reads from IgG antibody as the control. SPI1 over-enrichment in the called peaks was confirmed with Simple Enrichment Analysis (SEA, v5.5.5). Overlapping peaks between replicates of the same condition were kept for further processing. The peaks were processed with multiBamSummary function from deepTools to obtain counts matrix per sample for downstream analysis.
Differential peak enrichment analysis was run between conditions (MUT vs WT and IFN⍺-treated vs control) using DESEQ2 for distal PU.1 peaks. Differential peaks in each condition were centered around SPI1 motif and motif enrichment was performed for regions +/- 250 bp from SPI1 motif using HOMER (v.4.1.1). Differential ranking between each motif two conditions was calculated.
Assembly: GRCh38
Supplementary files format and content: Processed 10X data is matrix table of gene/peak counts per cell for each sample
Supplementary files format and content: Output matrices processed from genotyping data have two columns: barcodes (BC) and Genotype
Library strategy: Ironthrone-GoT

value: IFNa

value: Ironthrone-GoT


title: CAL27,none,rep2


title: CAL27, FOXK1 CUT&RUN,rep2

description:
CAL27_FOXK1_rep_mean.bw
CAL27_FOXK1.bed

data_processing:
The sequencing reads were aligned to the hg19 genome with Bowtie2 with ‘--end-to-end --very-sensitive --no-mixed --no-discordant --phred33 -I 10 -X 700 –dovetail’ parameters.
Peak calling was performed with MCAS2 (p <0.05). These peaks were then subjected to IDR analysis to identify reproducible peaks with IDR cutoff of 0.05.
The final peak set was determined by combining the IDR filtered peaks and the common peaks between the two biological replicates.
Assembly: hg19
Supplementary files format and content: BigWig, Bed
Library strategy: CUT&RUN

extract_protocol:
CUT&RUN was performed on 0.4-1 millioncells using reagents from the EpiCypher kit (EpiCypher, 14-1048), either using against FOXK1 or IgG control.
Cells were washed with PBS, harvested with trypsin, quenched with DMEM, and resuspended in cold PBS + 0.1% BSA to be maintained afterwards at 4°C. Cells were washed twice with PBS + 0.1% BSA, and quantified with a Countess. Nuclei were extracted by resuspending in 100 μL nuclei extraction buffer per 1-2.5 M cells (20 mM HEPES pH 7.5, 10 mM NaCl, 0.5 mM spermidine, 0.1% BSA, 0.1% NP-40, and 1 tablet Roche mini protease inhibitor per 10-15 mL) and incubating for 10 minutes on ice. 5-10 volumes of wash buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM spermidine, 0.1% BSA, 0.05%Triton X-100, and Roche protease inhibitor) were added, cells centrifuged at 600 RCF, and supernatant removed. After an additional wash, cells were quantified using a Countess. 400K-1M nuclei were bound to 10 μL activated Concanavalin A beads (EpiCypher, 21-1401) per sample for 10 min incubation at 4°C. After removing supernatant, beads were incubated with rocking at 4°C for 2 hrs with antibody diluted in 50 μL Antibody Buffer (wash buffer with 2 mM EDTA). After incubation, cells were washed twice with wash buffer, and incubated with 1.5-2.5 μL pAG-MNase (EpiCypher, 15-1016) per sample for 1 hour at 4°C with rocking, then washed with once with 150 μL wash buffer, then 150 μL low salt buffer (3.5 mM HEPES pH 7.5, 0.5 mM spermidine, 0.1% BSA, 0.05%Triton X-100, and Roche protease inhibitor) and resuspend in 50 µL low salt buffer. After 5 minutes on ice, 2 μL of 250 mM CaCl2 was added to the beads and beads were subsequentially incubated on ice for 18-25 minutes. The reactions were quenched with an equal volume 2X STOP buffer (300 mM NaCl, 20 mM EDTA, 40 mM EGTA, 0.025% SDS, 0.5% Triton X100, 0.05 mg/mL RNAse A, 0.05 mg/mL glycogen) for 30 minutes at 37°C according to the manufacturer’s protocol. Beads were placed on a magnet, and supernatant containing released fragments was removed. DNA was extracted by a Zymo DNA Clean and Concentrator (Zymo Research, D4014), eluting in 27 μL water. Library was prepared using the NEB Ultra II DNA library prep kit (NEB, E7103S) following dx.doi.org/10.17504/protocols.io.bagaibse6, with the following modifications: the adapter was diluted 20-25 fold, PCR was performed with SYBR dye to visualize amplification, and after PCR, 20 μL of water, then 30 μL of beads (0.6X instead of 0.8X) beads were added for the initial selection to remove large small fragments, followed by the second size selection using the addition of 20 μL beads (1X instead of 1.2X) for library capture.


title: duodenum male high-dose exposure rep3

description: Library name: D_19

value: duodenum


title: CAL27, none ,rep1


title: IFN13_cd34pos_sn10x

description: 10x genomics

data_processing:
10x data was processed using Cell Ranger 1 and v6.1.2 using default parameters. RNA reads were aligned to the human referece genome.
Amplicon reads was processed following steps: 1) Identification of reads with proper priming, 2) Identification of cell barcodes (CB) that match with the whitelisted CB, 3) Replacement of CB that are not perfectly matched with the whitelist CB, 4) Deduplication of reads, 5) Analyze reads with CB that are also observed in the 10x RNA-seq dataATAC-seq 6) Collapse networks of closely related UMIs within CBs to correct for sequencing error, 7) Matching of CB/UMI pairs found in 10x scRNA-seq data, discarding those matching non-CALR genes and those matching no gene below a supporting read threshold.
CUT&RUN data were down-sampled to have same number of reads across all conditions within the sample replicate to account for difference in sequencing depth. Among the down-sampled reads, low-quality reads were filtered out using Trimmomatic, resulting reads were aligned with hg38 genome with Bowtie2, and PCR duplicates were removed with SAMtools with a previously described workflow. Aligned reads were then used for peak calling with MACS3 with a q-value threshold of 0.01 and reads from IgG antibody as the control. SPI1 over-enrichment in the called peaks was confirmed with Simple Enrichment Analysis (SEA, v5.5.5). Overlapping peaks between replicates of the same condition were kept for further processing. The peaks were processed with multiBamSummary function from deepTools to obtain counts matrix per sample for downstream analysis.
Differential peak enrichment analysis was run between conditions (MUT vs WT and IFN⍺-treated vs control) using DESEQ2 for distal PU.1 peaks. Differential peaks in each condition were centered around SPI1 motif and motif enrichment was performed for regions +/- 250 bp from SPI1 motif using HOMER (v.4.1.1). Differential ranking between each motif two conditions was calculated.
Assembly: GRCh38
Supplementary files format and content: Processed 10X data is matrix table of gene/peak counts per cell for each sample
Supplementary files format and content: Output matrices processed from genotyping data have two columns: barcodes (BC) and Genotype
Library strategy: GEX+ATAC

value: Bone marrow

value: GEX+ATAC


title: CAL27, FOXK1 CUT&RUN,rep1

description:
CAL27_FOXK1_rep_mean.bw
CAL27_FOXK1.bed


title: duodenum male high-dose exposure rep2

description: Library name: D_18


title: SCC13,p63 siRNA,rep2

value: p63 knockdown


title: IFN07_cd34pos_snGenotyping

description: Ironthrone-GoT

value: Essential thrombocythemia


title: CAL27,IgG CUT & RUN,rep2

description:
CAL27_FOXK1_rep_mean.bw
CAL27_FOXK1.bed

value: IgG


title: duodenum male high-dose exposure rep1

description: Library name: D_17

value: Sprague-Dawley

value: adult

value: Male-High Dose/50mg/kg


title: SCC13,p63 siRNA,rep1

value: Squamous cell carcinoma


title: IFN07_cd34pos_sn10x

description: 10x genomics

data_processing:
10x data was processed using Cell Ranger 1 and v6.1.2 using default parameters. RNA reads were aligned to the human referece genome.
Amplicon reads was processed following steps: 1) Identification of reads with proper priming, 2) Identification of cell barcodes (CB) that match with the whitelisted CB, 3) Replacement of CB that are not perfectly matched with the whitelist CB, 4) Deduplication of reads, 5) Analyze reads with CB that are also observed in the 10x RNA-seq dataATAC-seq 6) Collapse networks of closely related UMIs within CBs to correct for sequencing error, 7) Matching of CB/UMI pairs found in 10x scRNA-seq data, discarding those matching non-CALR genes and those matching no gene below a supporting read threshold.
CUT&RUN data were down-sampled to have same number of reads across all conditions within the sample replicate to account for difference in sequencing depth. Among the down-sampled reads, low-quality reads were filtered out using Trimmomatic, resulting reads were aligned with hg38 genome with Bowtie2, and PCR duplicates were removed with SAMtools with a previously described workflow. Aligned reads were then used for peak calling with MACS3 with a q-value threshold of 0.01 and reads from IgG antibody as the control. SPI1 over-enrichment in the called peaks was confirmed with Simple Enrichment Analysis (SEA, v5.5.5). Overlapping peaks between replicates of the same condition were kept for further processing. The peaks were processed with multiBamSummary function from deepTools to obtain counts matrix per sample for downstream analysis.
Differential peak enrichment analysis was run between conditions (MUT vs WT and IFN⍺-treated vs control) using DESEQ2 for distal PU.1 peaks. Differential peaks in each condition were centered around SPI1 motif and motif enrichment was performed for regions +/- 250 bp from SPI1 motif using HOMER (v.4.1.1). Differential ranking between each motif two conditions was calculated.
Assembly: GRCh38
Supplementary files format and content: Processed 10X data is matrix table of gene/peak counts per cell for each sample
Supplementary files format and content: Output matrices processed from genotyping data have two columns: barcodes (BC) and Genotype
Library strategy: GEX+HTO+ATAC

value: GEX+HTO+ATAC


title: duodenum male low-dose exposure rep8

description: Library name: D_16

value: Male-Low Dose/5mg/kg

value: Batch 2


title: CAL27, IgG CUT & RUN ,rep1

description:
CAL27_FOXK1_rep_mean.bw
CAL27_FOXK1.bed

value: WT


title: SCC13,control siRNA,rep2


title: IFN03_cd34pos_snGenotyping

description: Ironthrone-GoT


title: A66_Y_Zfp219_Ttran_3

first: Alex Chang-Yu

last: Chen

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

department: Krantz Family Center for Cancer Research

institute: Massachusetts General Hospital

description:
Library name: A66_Y_Zfp219_Ttran_3
Y_Zfp219_Ttran

data_processing:
Raw fastq files were downloaded from Illumina BaseSpace. Quality trimming and primer removal in raw fastq files were done with Trimmomatric (v.0.36) using the following parameters: LEADING:15 TRAILING:15 SLIDINGWINDOW:4:15 MINLEN:30 CROP:35. Trimmed reads were aligned to mm10 with Bowtie2 (v.2.2.9) using a maximum insert size of 1,000. Aligned bams were sorted, duplicates marked (Picard v.2.8.0) , and reads mapping to the blacklist region removed. Peak-calling using MACS (v.2.1.1) was performed on merged bam files (Samtools v. 1.3.1) from biological replicates using a q-value threshold of 0.001. Consensus peaks from all biological conditions were merged to create a single peak universe. Cut sites were extracted from each biological replicate and the number of cuts in each peak region was quantified to generate a raw counts matrix. Normalization and differential expression were performed using DESeq2 (v 1.28.1).
Assembly: mm10
Supplementary files format and content: tab-delimited text file including DESeq2 normalized counts

source_name: B16-OVA Tumor

extract_protocol:
DNA was purified using a QIAgen MinElute Reaction Cleanup kit
Post-PCR bead cleanup was done using Agencourt AMPure XP bead cleanup (Beckman Coulter/Agencourt), and library quality was verified using Tapestation analysis.

value: B16-OVA Tumor

value: Cas9 OT-1 CD8+ T cells

tag: gene knockout

tag: cell state

value: transitory, effector-like


title: duodenum male low-dose exposure rep7

description: Library name: D_15

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

value: duodenum

value: Batch 3


title: A66_Y_Zfp219_Ttran_2

description:
Library name: A66_Y_Zfp219_Ttran_2
Y_Zfp219_Ttran


title: duodenum male low-dose exposure rep6

description: Library name: D_14


title: IFN03_cd34pos_sn10x

description: 10x genomics

value: Bone marrow

value: IFNa


title: SCC13,control siRNA,rep1


title: IFN01_cd34pos_snGenotyping

description: Ironthrone-GoT

value: Essential thrombocythemia

value: Ironthrone-GoT


title: CAL27,p63 siRNA,rep2

value: p63 knockdown


title: A66_Y_Zfp219_Ttran_1

description:
Library name: A66_Y_Zfp219_Ttran_1
Y_Zfp219_Ttran


title: duodenum male low-dose exposure rep5

description: Library name: D_13

value: Sprague-Dawley

value: adult


title: CAL27,p63 siRNA,rep1

value: Squamous cell carcinoma


title: IFN01_cd34pos_sn10x

description: 10x genomics

value: GEX+HTO+ATAC


title: A66_Y_Zfp219_Tterm_3

description:
Library name: A66_Y_Zfp219_Tterm_3
Y_Zfp219_Tterm

value: terminally exhausted


title: duodenum male low-dose exposure rep4

description: Library name: D_12

value: Male-Low Dose/5mg/kg

value: Batch 2


title: CAL27,control siRNA,rep2

value: WT