title: Casp8_Tnfr1_dko_rep3

city: South San Francisco

first: Rohit

last: Reja

address: 1 DNA Way

institute: Genentech

data_processing:
GSNAP (version 2013-11-10) was used to align raw FASTQ reads to the mouse reference genome (GRCm38/mm10) with following parameters “-M 2 -n 10 -B 2 -i 1 -N 1 -w 200000 -E 1 --pairmax-rna=200000 --clip-overlap”. Reads were filtered to include only the uniquely mapped reads.
Assembly: GRCm38/mm10
Supplementary files format and content: tab-delimited text files include raw counts for each sample

source_name: Liver

extract_protocol:
Total RNA was isolated from cells and purified using RNeasy mini kits and on-column DNAse digestion per manufacturer (Qiagen) protocol. 0.5ug used for library preparation with a TruSeq RNA Sample Preparation Kit v2 (Illumina). The libraries were multiplexed and then sequenced on a HiSeq4000 (Illumina) to generate 30 million single end 50 bp reads.
RNA libraries were prepared for sequencing using standard Illumina protocols

value: Liver

value: Casp8 TNFR1 dKO

tag: developmental stage

value: No treatment


title: NaraCOVID_TP5_CD8_019_TCR

status: Public on Apr 01 2024

type: SRA

city: Noda

first: Hiroyasu

last: Aoki

state: Chiba

address: 2669 Yamazaki

department: Research Institute for Biomedical Sciences

country: Japan

institute: Tokyo University of Science

library_source: transcriptomic

data_processing:
Data processing of Bulk TCRseq was performed as described in Aoki et al. (Aoki et al., 2021).
Adapter trimming and quality filtering of sequencing data were performed using Cutadapt-3.2 and PRINSEQ-0.20.4. Sequencing data were processed by MiXCR-3.0.5. In MiXCR, filtered reads were aligned to reference mouse TCR V/D/J sequences registered in the international ImMunoGeneTics (IMGT) information system. The Variable (V) and Joining (J) segments of TCRs were represented in IMGT gene nomenclature. T-cell clones were determined as TCR reads with the same TCR V segment, J segment, and CDR3 nucleotide sequence.
Inter-sample contamination within samples using same UDIs were excluded by "Decontaminate" function of vdjtools with the parameter "-r 10".
For bulk TCRseq data, we regarded the CD4+-CD8+ overlapping clones in the same participant as contamination during T-cell isolation from PBMC because CD4+ and CD8+ T-cell repertoire are mutually exclusive (Aoki 2021). Thus, these overlapping clones were categorized into either CD4+ or CD8+ according to their dominance in CD4+ and CD8+ repertoire using VDJtools-1.2.1. The sequencing reads of bulk TCR-seq samples were normalized to 100,000 reads by the “DownSample” command of VDJtools-1.2.1. To preserve the minor clonotypes, we performed the normalization ten times and pooled these down-sampled repertoire data for each sample, by “JoinSamples” command of VDJtools-1.2.1.
Assembly: IMGT reference sequences of human TRA and TRB
Supplementary files format and content: Text files in VDJtools format include read count and frequency, complementarity determining region 3 (CDR3) nucleotide and amino acid sequences, Variable(V)/ Diversity(D)/ Joining(J) segment names, and V, D and J segment boundaries within CDR3 nucleotide sequence of individual clones
Library strategy: Bulk TCR-seq

molecule: total RNA

organism: Homo sapiens

source_name: Peripheral blood T cells

extract_protocol:
PBMCs were isolated from whole blood with BD vacutainer CPT tubes (BD), resuspended in CELLBANKER 1 (Takara), and stored in -80°C.
CD4+ and CD8+ T cells were magnetically isolated from PBMC. PBMC were thawed by warming frozen cryovials in a 37°C water bath. Cells were stained for 20 minutes at 4°C with 1st antibody mix (Antibody Mix 1 in Supplementary Table S1). After washing with MACS buffer [0.5% BSA (Nacalai), 2 mmol/L EDTA (NIPPON GENE) in PBS (Nacalai)], Cells were stained for 20 minutes at 4°C with 2nd antibody mix; BD IMag anti-PE Magnetic Particles-DM (Clone E31-1459, BD) and anti-APC-Biotin (clone APC003, BioLegend). Then, the cell suspension was diluted with MACS buffer and set on a magnet plate (DynaMag96SideSkirted, Veritas). The positive fraction was collected as CD4+ T cells. The negative fraction was stained with BD IMag Streptavidin Particles Plus-DM (BD) for 20 minutes at 4°C. Then, the cell suspension was diluted with MACS buffer and set on a magnet plate. The positive fraction was collected as CD8+ T cells and B cells. The counts of CD4+ T cells, CD8+ T cells, and B cells were determined using Flow-Count fluorospheres (Beckman) and a Cytoflex flow cytometer (Beckman) and FlowJo software (version 10.8.1; BD Biosciences).
Bulk TCR sequencing library was prepared according to the previous report (Aoki et al., 2023).  In brief, mRNA in T-cell lysates were captured by incubating with 10 μL of Dynabeads M270 streptavidin (Thermo Fisher Scientific) bound to 2.5 pmol of BioEcoP-dT25-adapter primers for 30 minutes at room temperature under gentle rotation. mRNA-trapped beads were suspended in 10 µL of RT mix and incubated for 60 min at 42ºC to perform reverse transcription (RT) and template-switching. The composition of the RT mix was as follows; 1× First Strand buffer (Thermo Fisher Scientific), 1 mM dNTP (Roche), 2.5 mM DTT (Thermo Fisher Scientific), 1 M betaine (Sigma-Aldrich), 9 mM MgCl2 (NIPPON GENE), 1 U/µL RNaseIn Plus RNase Inhibitor (Promega), 10 U/µL Superscript II (Thermo Fisher Scientific), and 1 µM of i5-TSO. To amplify the TCR cDNA containing complementarity determining region 3 (CDR3), nested PCR of the TCR locus was performed using KAPA Hifi Hotstart ReadyMix (KAPA Biosystems), followed by the purification of PCR product using an AM Pure XP kit (Beckman Coulter) at a 0.7:1 beads-to-sample ratio. The third PCR was performed to amplify TCR libraries and add adaptor sequences for the next-generation sequencer. The third-PCR products were purified as second PCR. Final TCR libraries, whose lengths were about 600 base pairs, were quantified using a KAPA SYBR Fast qPCR Kit (KAPA Biosystems), and size distribution was analyzed by Microchip Electrophoresis System MultiNA (Shimadzu). TCR libraries were sequenced using an Illumina Novaseq 6000 S4 flowcell (67 bp read 1 and 140 bp read 2) (Illumina) using NovaSeq 6000 S4 Reagent Kit v1.5 (200 cycles, Illumina). Only read2 contained the sequence regarding the definition of T-cell clones. Oligonucleotides and reaction conditions for library preparation were summarized in Table S2 and S3, respectively.

treatment_protocol: 38 healthcare workers (over 20 years old) at Nara Medical University in Japan received two doses of Pfizer-BNT162b2 vaccine between 10 March and 30 March 2021.

tag: treatment

value: Three shots of Pfizer-BNT162b2 vaccine

tag: cell type

value: CD8+ T cells and CD19+ B cells

tag: age

tag: Sex

value: female

tag: date

value: 3 month after receiving the second shot


title: VUMC0437

type: RNA

city: Nashville

first: Cong

last: Wang

state: TN

address: 2525 West End Ave

country: USA

institute: Vanderbilt University

hyb_protocol: NanoString nCounter miRNA Expression Assay manufacturer's protocol

data_processing: miRNAs were normalized in the following steps: 1) background correction to remove non-specific binding. The background level was calculated as an aggregate summary of seven negative control probes with no target transcript. Mean of the calculated background level for each sample was then subtracted from the adjusted probe value to remove background noise. 2) Normalization of miRNA counts using mean counts of three spike-in synthetic control miRNAs (ath-miR159a, cel-miR-248, and osa-miR414) to control for variances on the starting material and the miRNA purification efficiency. 3) Sample content normalization using geometric mean counts of the top 50 miRNAs expressed excluding hsa-miR-320e, hsa-miR-16-5p and hsa-miR-451a because these miRNAs may be related to red blood cell contamination or hemolysis. After these steps, the expression level of each miRNA was calculated.

label: NanoString

source_name: plasma

extract_protocol: Qiagen’s miRNeasy Plasma Kit manufacturer's protocol

tag: study site (plco=1 swmhs=2 sccs=3 mec=4)

tag: case control status (control=0 case=1)

tag: sets

tag: sex (female=0 male=1)

tag: age at diagnosis

tag: race (white=1 black=2 other=3)


title: Inguinal White adipose tissue, Day14, scRNA-seq

city: Shanghai

first: Yeung

last: Sivannn

address: Dongan Road

country: China

institute: Fudan University

description: SAMPLES 2

library_source: transcriptomic single cell

data_processing:
Raw sequencing reads of the cDNA library were mapped to the reference genome by 10X Genomics Cell Ranger (v3.1.0). Genome Reference Consortium Mouse Build 38 (GRCm38) was used as reference for 10X Cell Ranger.
The cell barcodes in the file are designated by the suffixes -1, -2, -3, -4, and -5, representing samples 1, 2, 3, 4, and 5, respectively.
Assembly: mm10
Supplementary files format and content: Tab-separated values files and matrix files

organism: Mus musculus

source_name: Inguinal White adipose tissue

extract_protocol:
Stromal vascular fraction of mouse iWAT were prepared by enzymatic dissociation with collagenase Ⅷ (sigma c2139).
Cells were loaded into Chromium microfluidic chips and barcoded within a 10× Chromium Controller (10X Genomics), Single cell transcriptome were reverse-transcribed into cDNA library containing 10X cell barcodes and unique molecular identifiers (UMI) information. All procedures were performed with reagents from Chromium Next GEM Single Cell 3ʹ GEM, Library & Gel Bead Kit v3.1 (10X Genomics, Cat. No. 1000121), following the manufacturers’ protocol. All the libraries were sequenced in a PE150 mode (Pair-End for 150bp read) on the NovaSeq platform (Illumina).

tag: tissue

value: Inguinal White adipose tissue

value: 14 days old


title: NaraCOVID_TP5_CD8_017_TCR

value: male


title: NaraCOVID_TP5_CD8_018_TCR


title: Casp8_Tnfr1_dko_rep1

state: CA

tag: genotype


title: Inguinal White adipose tissue, Day7, scRNA-seq

description: SAMPLES 1

value: 7 days old


title: VUMC0444


title: CARD14E138A_3hr_1

city: London

first: Tolar

last: Pavel

address: Gower St

country: United Kingdom

institute: UCL

data_processing:
The RSEM package (version 1.3.0) in conjunction with the STAR alignment algorithm (version 2.5.2)  was used for the mapping and subsequent gene-level counting of the sequenced reads with respect to Ensembl GRCm38-release-89 transcriptome
Differential expression analysis was performed with the DESeq2 package (version 1.10.1) within the R programming environment (version 3.3.2)
Assembly: GRCm38
Supplementary files format and content: Tab delimited text files containing TMM normalised data. Rows represent ensembl gene identifiers and columns represent individual samples.

source_name: CARD14E138A-3xFLAG HaCaT-TR

growth_protocol: Cells are at least two doublings from liquid nitrogen thaw and never allowed to be in prolonged culture with high confluency

extract_protocol:
RNA was extracted with and RNEasy kit (Qiagen). A fraction of each sample was taken and assessed for RNA quality. mRNA was then purified using a poly-A KAPA mRNA Hyper Prep kit (Roche)
Samples were processed with the KAPA mRNA HyperPrep kit, according to manufacturer’s instructions, which includes: End repair and A-tailing, which produces end-repaired, 5′-phosphorylated, 3′-dA-tailed, dsDNA fragments. Adapter ligation, during which dsDNA adapters with 3′-dTMP overhangs are ligated to 3′-dA-tailed library fragments. Library amplification, which employs high-fidelity, low-bias PCR to amplify library fragments carrying appropriate adapter sequences on both ends.

treatment_protocol: Cells were induced with tetracycline as part of the Tet-on (Trex) system to cause gene induction of CARD14 WT or mutant

value: Skin cell line

value: keratinocyte

value: +Tetracycline for indicated timepoint

tag: environmental condition

value: 37C, 5% CO2 gas


title: NaraCOVID_TP5_CD8_016_TCR


title: VUMC0435


title: exGF_9_L

city: Frederiksberg C

first: Camilla

middle: Hartmann Friis

last: Hansen

address: Ridebanevej 9, 2nd floor

department: Faculty of Health Sciences

country: Denmark

institute: University of Copenhagen

data_processing:
Illumina CASAVA software used for basecalling.
Adaptors trimmed with Fastp, filtered for low quality sequence and mapped to Mus Musculus(GRCm38/mm10) genome with HISAT2 v 2.0.5
Gene count file made using FeatureCounts v 1.5.0
Assembly: mm10
Supplementary files format and content: matrix table with raw gene counts for every gene and every sample

molecule: polyA RNA

source_name: liver

extract_protocol:
Rneasy Lipid Mini kit
RNA libraries were prepared for sequencing using standard Illumina protocols

value: liver

tag: strain

value: Swiss Webster

value: Male


title: VUMC0434


title: VUMC0433


title: Casp8_Tnfr1_dko_rep2

value: Liver

value: Casp8 TNFR1 dKO


title: NaraCOVID_TP5_CD8_015_TCR

value: Three shots of Pfizer-BNT162b2 vaccine


title: Casp8_Mlkl_dko_rep3

value: Casp8 MLKL dKO

value: No treatment


title: CARD14E138A_0hr_1


title: exGF_7_L


title: exGF_6_L


title: Casp8_Mlkl_dko_rep1


title: VUMC0436


title: NaraCOVID_TP5_CD8_013_TCR

value: CD8+ T cells and CD19+ B cells

value: female

value: 3 month after receiving the second shot


title: VUMC0431


title: NaraCOVID_TP5_CD8_012_TCR

value: male


title: Tnfr1ko_rep2


title: exGF_5_L


title: VUMC0430


title: NaraCOVID_TP5_CD8_011_TCR


title: SPF_5_L

value: SPF


title: NaraCOVID_TP5_CD8_010_TCR


title: SPF_4_L


title: WT_rep3

value: WT


title: WT_rep2


title: NaraCOVID_TP5_CD8_014_TCR


title: Casp8_Mlkl_dko_rep2


title: NaraCOVID_TP5_CD8_007_TCR


title: NaraCOVID_TP5_CD8_008_TCR


title: VUMC0429


title: SPF_3_L


title: SPF_2_L


title: VUMC0427


title: VUMC0426


title: Tnfr1ko_rep1


title: SPF_1_L


title: exGF_8_L


title: AOM/DSS advanced tumor (w13) TANs 2

city: Chicago

first: Ronen

last: Sumagin

state: IL

address: 303 E Chicago Ave

department: Pathology

institute: Northwestern University

data_processing:
Read alignment with STAR. Raw reads were de-multiplexed into FASTQ format using bcl2fastq software (Illumina) The FASTQ files were quality checked for called bases of each read. Pre-processing of raw data was performed by Trimmomatic option in Chipster where reads of quality below 20 were eliminated. The Illumina adapters were removed from the 3’ ends by cutadapt command-line program that operated under two adapter trimming algorithms 71.
Trimmed reads were aligned to the Mus musculus genome mm10 and human genome hg38 using STAR RNA-seq aligner (version STAR_2.5.1 72). Of note, pre-processing of the reads was performed in conjunction with the mm10 mouse gene annotation file obtained from Ensembl (http://useast.ensembl.org/index. html). Reads with read lengths <35 were discarded. The read counts for individual genes were generated by htseq-count script derived from the HTseq framework v.0.6.1p1 that counts and calculates the read overlaps of genes within the mouse and human genome.
To identify DEGs, normalization for sequencing depth followed by variance-stabilizing transformation as well as differential expression calculations were performed via DESeq2 R Bioconductor package 1.16.1, which employed the Wald test to shrink Log-Fold-Changes (LFC) and their standard errors.  The cut-off for determining significantly differentially expressed genes was determined with a stringency level where an FDR-adjusted p<0.05 and |log2FC| <1 using the Benjamini-Hochberg method. In addition, genes with normalized counts < 100 were removed and not considered for future analysis. To execute principal-component analysis (PCA) and heatmap visualization, read counts were transformed to log2(count per million) and corrected for batch effects using limma package. In these analyses, PCA plots were made using the pca() function and hierarchical clustering with the dist() function.
Assembly: hg18 (PDX data) and mm3 (neutrophil AOM/DSS data)
Supplementary files format and content: count matrices
Supplementary files format and content: GSEA, GO, and DEG outputs

source_name: advanced CRC tumors

extract_protocol:
For sorting of PMNs or other myeloid cells, we utilized Lyz2EGFP reporter mice with additional staining for Ly6G, Ly6C, CD11b, CD45, and CD64. FACS was performed on a 3-laser sorter FACS Melody and a 6-color laser sorter FACS Aria at the Northwestern Flow Cytometry core. EpCAM+ tumor or epithelial cells as well as Ly6Gneg CD64hi¬¬ macrophages were gated out to avoid RNA contamination from other cell types. CD45+ CD11b+ Ly6Cint Lyz2EGFP Ly6Ghi/int were defined as PMNs/ TANs and sorted directly into RNA Lysis Buffer (RLT plus buffer, Qiagen), supplemented with excess amount of RNaseOUT (10 U/μL, Invitrogen). RNA from PMNs was immediately extracted using the RNeasy Mini Plus Kit (Qiagen) and subjected to RNA integrity (RIN) analysis. For quality control of PMN cell sorting, additional replicates were performed with identical gating strategy and subjected to either more rigorous flow cytometric analyses using additional PMN markers, or using Cytospin and immunofluorescence for imaging. Enrichment of PMN gene sets was performed following RNA sequencing to confirm the transcriptomic identity of the sorted cells as well as validity of downstream bioinformatic analyses
The NEBNext Ultra II RNA Library Prep Kit for Illumina (New England Biolabs, Ipswich, MA) was used for library preparation per manufacturer’s protocol with the addition of NEBNext rRNA depletion option. Following fragmentation to 200-500 base pairs (bp) by Bioruptor Sonicator (Diagenode), reverse transcription (RT) was performed to convert RNA to cDNA, followed by end-repair, adaptor ligation, and PCR amplification of cDNA libraries per manufacturer’s instruction (New England Biolabs, Ipswich, MA). Prior to sequencing, prepared libraries were assessed for fragment sizing on a Bioanalyzer using High Sensitivity DNA chip and re-quantified with Qubit. Library sequencing was performed on an Illumina HiSeq 4000 NGS System (Illumina, San Diego, CA) to generate 50 bp reads with 300 million reads and 30x read depth.

treatment_protocol: To initiate carcinogenesis, mice were given a single dose of azoxymethane (AOM) by intraperitoneal (i.p.) injection (12.5 mg/kg body weight). A week after, mice were treated with up to 5 cycles of DSS 3% in drinking water (1 cycle = 5 days of DSS + 14 days recovery). Immediately after sacrifice, peripheral blood was drawn from the vena cava; bone marrows from both hind-legs (femur and tibia) were collected; spleen and tumor-bearing colons were extracted for phenotypic evaluations and downstream molecular/functional assays. For PDX experiments, a total of 15 PDX-bearing mice were purchased from Jackson Laboratory (Bar Harbor). PDXs were established by the Jackson Labs as follows; metastatic stage IV colon adenocarcinoma with BRAFV600E and PI3KCAG1049R mutations derived from a 30-year-old male patient (Colon Cancer, PDX Model TM00170) were subcutaneously grafted (2x106 tumor cells/mouse in 40μL PBS) into flanks of NOD.Cg-Prkdcscid Il2rgtm-1Wjl/SzJ NSG mice aged 7-8 weeks (strain 005557 NSG). Through the duration of the experiments, tumor diameters were measured bi-weekly by calipers. Once tumors reached 500mm3, mice were randomly divided into 3 treatment groups (Control, Anti-OPN, and NSC405020. Anti-OPN (clone 103D6) was administered at 5.0mg/kg/mice, daily, i.p.) while NSC 405020 was administered 1.0mg/kg/mice (3 times per week, i.p.) for 21 days (endpoint).

value: advanced CRC tumors

value: primary immune:neutrophils

value: AOM/DSS


title: VUMC0424


title: NaraCOVID_TP5_CD8_005_TCR


title: NaraCOVID_TP5_CD8_004_TCR


title: WT_rep1


title: Taurine-3

city: Xi'an

first: Xiaodi

last: Zhao

address: No.127 Changle West Road

department: Xijing Hospital of Digestive Diseases

institute: Xijing Hospital of Fourth Military Medical University

data_processing:
The 10x Genomics Cell Ranger (7.1.0 version) pipeline was used to demultiplex raw files into FASTQ files, extract barcodes and UMI, filter, and map reads to the corresponding reference genome, and generate a matrix containing UMI counts.
A 10x Genomics Cell Ranger pipeline was used to identify clonotypes by alignment and annotation with the default settings.
Assembly: mm10
Supplementary files format and content: Tab-separated values files and matrix files

source_name: Tumor

extract_protocol:
The sorted immune cell (CD45+) suspension form mice tumors were loaded into Chromium microfluidic chips with 30 (v3) chemistry and barcoded with a 10× Chromium Controller (10X Genomics). RNA from the barcoded cells was subsequently reverse-transcribed and sequencing libraries constructed with reagents from a Chromium Single Cell v3 reagent kit (10X Genomics) according to the manufacturer’s instructions. Sequencing was performed with Illumina (NovaSeq 6000) according to the manufacturer’s instructions (Illumina).
Raw reads were demultiplexed and mapped to the reference genome by 10X Genomics Cell Ranger pipeline (https://support.10xgenomics.com/single-cell-gene- expression/software/pipelines/latest/what-is-cell-ranger) using default parameters. Then, downstream single-cell analyses were performed using Seurat package.
In brief, for each gene and each cell barcode (filtered by Cell Ranger), unique molecule identifiers were counted to construct digital expression matrices. To filter out low-quality cells, we set the following criteria: 500 ≤ detected genes number ≤ 8000; mitochondrial RNA ≤ 10%; non-blood red cell genes detected in ≤ 5% of all cells.

value: Tumor

tag: cell line

value: immune cell

value: 615 mice

value: Taurine


title: VUMC0432


title: AOM/DSS advanced tumor (w13) TANs 1


title: VUMC0428


title: Taurine-2


title: AOM/DSS early tumor (w8) TANs 2

source_name: early CRC tumors

value: early CRC tumors


title: VUMC0423


title: NaraCOVID_TP5_CD8_002_TCR

value: Three shots of Pfizer-BNT162b2 vaccine


title: NaraCOVID_TP5_CD8_003_TCR


title: NaraCOVID_TP5_CD8_001_TCR


title: AOM/DSS early tumor (w8) TANs 1


title: Saline-3

value: Saline


title: AOM/DSS advanced tumor (w13) blood neutrophils/PMNs 2

source_name: peripheral blood PBMC

value: peripheral blood PBMC


title: VUMC0420


title: VUMC0422


title: Taurine-1


title: NaraCOVID_TP5_CD8_006_TCR

value: CD8+ T cells and CD19+ B cells


title: Saline-2


title: AOM/DSS advanced tumor (w13) blood neutrophils/PMNs 1


title: NaraCOVID_TP5_CD4_041_TCR

value: CD4+ T cells


title: Saline-1


title: VUMC0419


title: AOM/DSS early tumor (w8) blood neutrophils/PMNs 2


title: VUMC0425


title: VUMC0417


title: VUMC0418


title: AOM/DSS early tumor (w8) blood neutrophils/PMNs 1


title: F32, recovery sleep, female, rep4

city: Philadelphia

first: Allan

middle: I

last: Pack

address: 125 South 31st St

institute: University of Pennsylvania

description: 10x Genomics

data_processing:
Data were processed using the Cellranger pipeline (10x genomics, v.6.1.2) for demultiplexing and alignment of sequencing reads to the mm10 transcriptome with the “include introns” parameter and created the feature-barcode matrices.
Assembly: mm10
Supplementary files format and content: Tab-separated value files and matrix files

source_name: brain

extract_protocol:
Nuclei were isolated from the dissected POA tissues using Minute Single Nucleus Isolation Kit for Neuronal Tissues/Cells (Invent Biotechnologies, Inc., BN-020) following manufacturer’s protocol with minor modifications. Specifically, Protector RNase inhibitor (Sigma, PN-3335399001) was added at a concentration of 0.2U/µl to all the buffers provided by the kit.
libraries were prepared using the 10x Genomics Chromium Single Cell 3’ Reagent kit v3 per manufacturer’s instructions. Libraries were uniquely indexed using the Chromium dual Index Kit, pooled, and sequenced on an Illumina NovaSeq 6000 sequencer in a paired-end, dual indexing run.

value: brain

value: tissue

value: RS


title: F29, recovery sleep, female, rep5

description: 10x Genomics


title: NaraCOVID_TP5_CD4_039_TCR

value: male

value: 3 month after receiving the second shot


title: DSS colitis colonic neutrophils/PMNs 1

source_name: inflamed colon

value: inflamed colon

value: primary immune:neutrophils


title: F40, recovery sleep, female, rep3

description: 10x Genomics


title: DSS colitis colonic neutrophils/PMNs 2

value: AOM/DSS


title: VUMC0416


title: NaraCOVID_TP5_CD4_038_TCR


title: VUMC0415


title: NaraCOVID_TP5_CD4_037_TCR

value: female


title: NaraCOVID_TP5_CD4_035_TCR


title: DSS colitis blood neutrophils/PMNs 1


title: F42, recovery sleep, female, rep2

description: 10x Genomics


title: NaraCOVID_TP5_CD4_040_TCR


title: F43, recovery sleep, female, rep1

description: 10x Genomics


title: Healthy blood neutrophils/PMNs 2


title: M35, recovery sleep, male, rep5

description: 10x Genomics


title: NaraCOVID_TP5_CD4_036_TCR


title: VUMC0414


title: DSS colitis blood neutrophils/PMNs 2


title: VUMC0413


title: Healthy blood neutrophils/PMNs 1


title: NaraCOVID_TP5_CD4_033_TCR


title: VUMC0411


title: NaraCOVID_TP5_CD4_032_TCR


title: THP7

city: Vienna

first: Tibor

last: Harkany

address: Spitalgasse 4

department: Department of Molecular Neurosciences (Center for Brain Research)

country: Austria

institute: Medical University of Vienna

data_processing:
Pre-processing
The Cell Ranger pipeline (v7.1.0) (Zheng et al. 2017) was used to perform sample demultiplexing, barcode processing and single-nuclei gene counting. Briefly, samples were demultiplexed to produce a pair of FASTQ files for each sample. Reads containing sequence information were aligned using the optimised mouse genome reference (vmm10_optimized_v.1.0) provided by Pool’s lab based on the default Cell Ranger mm10 genome version 2020-A that was cleared from gene overlaps, poorly annotated exons and 3’-UTRs and intergenic fragments (Pool et al. 2022). PCR duplicates were removed by selecting unique combinations of cell barcodes, unique molecular identifiers (UMIs) and gene ids with the final results being a gene expression matrix that was used for further analysis.
Quality control
Droplet selection
The droplet selection method of Cell Ranger identified 923 nuclei in ventrobasal thalamus and in principal sensory trigeminal nucleus - 731 nuclei based on EmptyDrops method (Lun et al. 2019) incorporated into cellranger count pipeline.
Ambient RNA removal
Using those values as the expected number of cells, we applied a neural network-based approach called CellBender (docker://etretiakov/cellbender:v0.0.1) (Fleming et al. 2022). We set a false positive rate threshold at the level of 0.01 and set the neural network to learn over 150 epochs with a total of 5000 droplets included based on knee plots (please see online supplementary Cell Ranger reports).
Doublets detection
We quantified log probability to be a doublet for every cell based on apriori knowledge of genotypes from each input sample and called variant occurrence frequencies that allowed to cluster nuclei to source organism or classify as a doublet (Heaton et al. 2020) (see shub://wheaton5/souporcell).
Further filtering
Gene annotation information was added using the gprofiler2 package (v0.2.1) (Reimand et al. 2016); thus we filter cells based on high content of mitochondrial, ribosomal or hemoglobin proteins genes, specifically thresholds set as 1%, 1% and 0.5%; additionally pseudogenes and poorly annotated genes were also deleted from count matrix. Moreover, cells of low complexity were filtered out as (log10Genes/log10UMI<0.8). Therefore, cells were assigned cell cycle scores using the CellCycleScoring function in the Seurat package (v4.3.0).
Clustering
Gene selection
We used the selection method in the Seurat package (v4.3.0) (Satija et al. 2015; Stuart and Satija 2019), which uses a modern variance stabilising transformation statistical technic that utilises scaling to person residuals (Hafemeister and Satija 2019). That way, we selected 3000 highly variable genes per dataset and regressed out complexity and cell-cycle variability prior to the final scaling of filtered matrixes.
Graph-based and multi-level reconcile tree clustering
We performed Leiden algorithm graph-based clustering. PCA was performed using the selected genes and the jackknife tested (Chung and Storey 2015) principal components (we tested the significance of feature for randomly picked 1% of data over 1000 iterations; see PCScore function in functions.R script of code directory) were used to construct a shared nearest neighbour graph using the overlap between the 15 nearest neighbours of each cell. Leiden modularity optimisation (Traag, Waltman, and Eck 2019) was used to partition this graph with an array of resolution parameters where 30 modularity events were sampled between 0.2 and 2.5. Clustering tree visualisations (Zappia and Oshlack 2018) were produced using the clustree package (v0.5.0) showing the resolution of previously identified clusters. By inspecting these resolutions reconcile tree produced by mrtree package (v0.0.0.9000) (Peng et al. 2021) and calculating adjusted multi-resolution Rand index chosen as maximum value if there is no higher modularity within 0.05 AMRI difference (see SelectResolution in function.R file of code directory).
Marker genes
Marker genes for each cluster were identified using logreg test (Ntranos et al. 2019) implemented in Seurat framework (v) (Stuart and Satija 2019). Genes were considered significant markers for a cluster if they had an FDR less than 0.001. Identities were assigned to each cluster by comparing the detected genes to previously published markers and our own validation experiments.
Assembly: UCSC mm10 (GRCm38)
Supplementary files format and content: h5Seurat R data files include Seurat 4 object for each Sample

source_name: Ventrobasal thalamus

extract_protocol:
Seven-day old mice (n = 3, C57BL/6JRj of mixed sex) were perfused with ice-cold modified HEPES solution containing (in mM): 90 NaCl, 3 KCl, 1.2 HEPES, 20 Na-pyruvate, 3 Na-ascorbate, 5 NaHCO3, 30 D-glucose, 25 MgSO4, 2 CaCl2, 1.5 L-glutathione; the brains dissected, and sliced at 400 µm thickness on a vibratome (settings: speed 0.3, vibration 1.4; Leica VT1200). The ventrobasal thalamus and principal trigeminal sensory nucleus were simultaneously punched out under a stereomicroscope and collected in separate cryovials. The samples were snap frozen using isopentane to minimize the formation of ice crystals and to preserve tissue integrity. Samples were kept on -80˚C until RNA extraction, single-nucleus library preparation, and RNA-sequencing.
For single-nucleus RNA-seq, nuclei were isolated from snap-frozen tissues by direct dissociation in cold Nuclei EZ Lysis buffer (Sigma # NUC101) using a pestle. The lysate was incubated on ice for 10 min, filtered through 70 µm-strainer meshes, and centrifuged at 500 rcf at 4˚C for 5 min. Pelleted nuclei were washed in ‘nuclei wash and resuspension buffer’ [1% BSA in 1x DPBS plus 0.5 U/µl Protector RNase-Inhibitor (Sigma #3335402001)], followed by centrifugation at 500 rcf at 4˚C for 5 min. Pelleted nuclei were then washed 3x before taking it up in nuclei wash and resuspension buffer supplemented with 7-amino-actinomycin D (7-AAD, BioLegend # 420404). After straining through a 40-µm mesh, single nuclei were sorted using a SONY SH800S cell sorter. Sorted nuclei were processed for single nuclei RNA-seq using the Chromium Next GEM Single Cell 3' Reagent Kit v3.1 (10x Genomics #PN1000128) according to manufactures instructions.

value: wild type

value: Ventrobasal thalamus

tag: libbatch


title: VUMC0410


title: NaraCOVID_TP5_CD4_031_TCR

tag: treatment


title: M7, recovery sleep, male, rep2

description: 10x Genomics

tag: cell type


title: M6, recovery sleep, male, rep3

description: 10x Genomics


title: M5, recovery sleep, male, rep4

description: 10x Genomics


title: NaraCOVID_TP5_CD4_030_TCR

tag: age


title: VUMC0408


title: TBK1-cKD ∆IKKe + R837 8h [SAM24408592]

data_processing:
Reads were aligned to the GRCm38 genome using GSNAP and genes were counted using the HTSeqGenie Bioconductor package. Gene models were from our internal database and available in the manuscript supplementary material. Supplementary files use Ensemble gene IDs, counts and RPKM statistics.
Assembly: GRCm38
Supplementary files format and content: tab-delimited text files include raw count and RPKM values for each sample

source_name: Bone Marrow

extract_protocol:
Bone marrow cells from femurs and tibia of wild-type C57BL/6N mice were cultured in Roswell Park Memorial Institute (RPMI) medium  supplemented with 10% heat-inactivated fetal bovine serum, 2 mM glutamine, 50 Î¼M 2-mercaptoethanol (Gibco), 100 U/ml penicillin, and 100 Î¼g/ml streptomycin (Gibco) and 50 ng/ml  of recombinant M-CSF (R&D Systems) on non-tissue culture treated 15 cm plates. On day 5 of culture, cells were harvested and 5 million cells were re-plated onto non-tissue culture  treated 10 cm plates in fresh media (same as that described above). After 2 additional days of culture, BMDMs from 3 individual mice (each corresponding to an independent biological replicate)  were harvested by scraping. Total RNA was isolated from cells and purified using RNeasy mini kits and on-column DNAse digestion per manufacturer (Qiagen) protocol.  0.5ug used for library preparation with a TruSeq RNA Sample Preparation Kit v2 (Illumina).  The libraries were multiplexed and then sequenced on a HiSeq4000 (Illumina) to generate 30 million  single end 50 bp reads.
RNA libraries were prepared for sequencing using standard Illumina protocols

treatment_protocol: Treatment with R837 for 8h

value: Bone Marrow

value: TBK1-cKD {delta}IKKe

value: Bone Marrow Derived Macrophages


title: VUMC0412


title: NaraCOVID_TP5_CD4_029_TCR

value: Three shots of Pfizer-BNT162b2 vaccine

tag: Sex


title: NaraCOVID_TP5_CD4_026_TCR

tag: date


title: NaraCOVID_TP5_CD4_027_TCR


title: TBK1-cKD ∆control + R837 8h [SAM24408591]

value: TBK1-cKD {delta}control


title: M8, recovery sleep, male, rep1

description: 10x Genomics


title: F10, normal sleep, female, rep4

description: 10x Genomics

value: SS


title: VUMC0406

tag: study site (plco=1 swmhs=2 sccs=3 mec=4)


title: F37, normal sleep, female, rep3

description: 10x Genomics

value: brain


title: TBK1-WT ∆IKKe + R837 8h [SAM24408590]

value: TBK1-WT {delta}IKKe


title: TBK1-WT ∆control + R837 8h [SAM24408589]

value: TBK1-WT {delta}control


title: VUMC0407

tag: case control status (control=0 case=1)


title: VUMC0405

tag: sets


title: F38, normal sleep, female, rep2

description: 10x Genomics

value: tissue


title: TBK1-cKD ∆IKKe [SAM24408588]


title: Pr5P7

source_name: Principal sensory trigeminal nucleus

value: Principal sensory trigeminal nucleus


title: NaraCOVID_TP5_CD4_025_TCR

value: CD4+ T cells

value: male

value: 3 month after receiving the second shot


title: F39, normal sleep, female, rep1

description: 10x Genomics


title: TBK1-cKD ∆control [SAM24408587]


title: NaraCOVID_TP5_CD4_024_TCR

value: female


title: VUMC0403

tag: sex (female=0 male=1)


title: M1, normal sleep, male, rep5

description: 10x Genomics


title: M17, normal sleep, male, rep4

description: 10x Genomics


title: TBK1-WT ∆IKKe [SAM24408586]


title: NaraCOVID_TP5_CD4_023_TCR


title: NaraCOVID_TP5_CD4_022_TCR


title: TBK1-WT ∆control [SAM24408585]


title: VUMC0404

tag: age at diagnosis


title: NaraCOVID_TP5_CD4_021_TCR


title: M33, normal sleep, male, rep2

description: 10x Genomics


title: VUMC0400

tag: race (white=1 black=2 other=3)


title: TBK1-cKD ∆control + R837 8h [SAM24408583]

tag: tissue


title: M18, normal sleep, male, rep3

description: 10x Genomics

tag: genotype


title: TBK1-cKD ∆IKKe + R837 8h [SAM24408584]


title: NaraCOVID_TP5_CD4_019_TCR


title: VUMC0399


title: db.db control diet

city: Otsu

first: Hiroaki

last: Tsuruta

state: Shiga

address: Tsukinowa

department: Department of Medicine

institute: Shiga University of Medical Science

description: !0x Genomics

data_processing:
The demultiplexing, barcooded processing, gene counting and aggregation were made usin Cell Ranger software v6.0.0
Assembly: mm10
Supplementary files format and content: Tab- separated values files and matrix files

source_name: Whole kideny

extract_protocol:
The flesh whole kidneys in each mice group were isolated respectively after perfused with DBPS from left ventricle. The extracted kidneys were immediately placed in an ice-cold DPBS environment. The kidneys were then meticulously cut into roughly 1 mm 3 cubes using a sterile surgical blade. The minced tissue was digested with 4mL of digestion buffer containing 3.0 mg/ml Collagenase type II, 1.5mg/ml Pronase E , 62.5 U/mL DNAse, and 5mM calcium chloride made up in DPBS(-). The digestion mixture was incubated at 37℃ in a water bath for 20 minutes. The remaining erythrocytes were removed with RBC lysis buffer. The debris and dead cell in the cells were removed with Debris removal solution kit, and Dead cell removal kit respectively. The cells were resuspended with PBS/BSA 0.04% after removing the debris and the dead cells.
Library was performed according to manufacters's instruncion (10x Genomics)

treatment_protocol: 8-week-old db/db mice were treated with fructose-free control diet or 60% fructose diet for 20 weeks. 8-week-old db/m mice were treated with control diet as db/db mice.

value: Whole kideny

value: Kidney cells

value: db/db

tag: diet

value: control


title: VUMC0398


title: db.db fructose diet

description: !0x Genomics

value: 60% fructose


title: NaraCOVID_TP5_CD4_018_TCR

value: Three shots of Pfizer-BNT162b2 vaccine


title: NaraCOVID_TP5_CD4_020_TCR


title: TBK1-WT ∆IKKe + R837 8h [SAM24408582]

value: Bone Marrow

value: Bone Marrow Derived Macrophages


title: TBK1-WT ∆control + R837 8h [SAM24408581]


title: NaraCOVID_TP5_CD4_017_TCR


title: TBK1-cKD ∆IKKe [SAM24408580]

value: TBK1-cKD {delta}IKKe


title: VUMC0397


title: VUMC0402


title: VUMC0396


title: db.m control diet

description: !0x Genomics

value: db/m


title: TBK1-cKD ∆control [SAM24408579]

value: TBK1-cKD {delta}control


title: VUMC0401


title: NaraCOVID_TP5_CD4_015_TCR


title: M34, normal sleep, male, rep1

description: 10x Genomics

value: brain

value: tissue

value: SS


title: NaraCOVID_TP5_CD4_016_TCR

value: CD4+ T cells


title: chronic 3

city: Takatsuki, Murasaki-cho

first: Toshiki

last: Yoshimizu

state: Osaka

institute: Japan Tobacco inc.

data_processing:
Raw reads, stored in fastq format, were filtered by removing the adaptor and low-quality reads for further analysis. To generate clean reads, inclusion of reads containing over 50% bases with quality greater than 5 and less than 10% “N” bases was allowed.
The clean reads were then mapped onto the mouse reference genome (version: GRCm38) using the STAR aligner (v2.6.0c).
The Feature Count (v0.6.1) program was used to get the gene level reads count based on the gene model annotation file downloaded from the Ensembl genome browser.
RNA library construction and sequencing analysis was performed by TaKaRa-Bio Co. Ltd.
Assembly: GRCm38
Supplementary files format and content: tab-delimited text file includes raw counts for each Sample
Supplementary files format and content: tab-delimited text file includes log2 CPM values for each Sample

source_name: lumbar spinal cord

extract_protocol:
Total RNA was extracted from the lumbar spinal cord with the RNeasy Lipid Tissue Mini kit (Qiagen Inc., Valencia, CA, USA) and RNA integrity was determined using the Agilent 2100 Bioanalyzer (Agilent Technologies, Inc., Palo Alto, CA, USA).
Two micrograms of total RNA per sample were used to construct sequencing libraries. Strand-specific RNA libraries were prepared using the TruSeq Stranded mRNA Library Prep Kit for Illumina (Illumina, Inc., San Diego, CA, USA).

value: lumbar spinal cord


title: TBK1-WT ∆IKKe [SAM24408578]

value: TBK1-WT {delta}IKKe


title: VUMC0394


title: chronic 2


title: VUMC0409


title: VUMC0393


title: NaraCOVID_TP5_CD4_013_TCR

value: female

value: 3 month after receiving the second shot


title: TBK1-WT ∆control [SAM24408577]

value: TBK1-WT {delta}control


title: relapse 2


title: TBK1-cKD ∆control + R837 8h [SAM24408575]


title: VUMC0392


title: TBK1-cKD ∆IKKe + R837 8h [SAM24408576]


title: relapse 3


title: NaraCOVID_TP5_CD4_014_TCR


title: NaraCOVID_TP5_CD4_012_TCR

value: male


title: NaraCOVID_TP5_CD4_011_TCR


title: TBK1-WT ∆IKKe + R837 8h [SAM24408574]


title: VUMC0390


title: TBK1-WT ∆control + R837 8h [SAM24408573]


title: NaraCOVID_TP5_CD4_010_TCR


title: VUMC0391


title: relapse 1


title: VUMC0395


title: remission 3


title: NaraCOVID_TP5_CD4_008_TCR


title: remission 2


title: VUMC0389


title: TBK1-cKD ∆IKKe [SAM24408572]


title: VUMC0388


title: NaraCOVID_TP5_CD4_007_TCR


title: remission 1


title: VUMC0387


title: TBK1-cKD ∆control [SAM24408571]


title: chronic 1


title: 1st peak 3


title: 1st peak 2


title: VUMC0386


title: TBK1-WT ∆control [SAM24408569]


title: NaraCOVID_TP5_CD4_005_TCR


title: TBK1-WT ∆IKKe [SAM24408570]


title: NaraCOVID_TP5_CD4_004_TCR


title: WT +R837 3h [SAM24402094]

treatment_protocol: Timecourse of treatment with R837 and/or Actinomycin D

value: WT


title: VUMC0385


title: VUMC0384


title: 1st peak 1


title: normal 2


title: WT +R837 3h [SAM24402095]


title: WT +R837 3h [SAM24402096]


title: NaraCOVID_TP5_CD4_002_TCR

value: Three shots of Pfizer-BNT162b2 vaccine


title: normal 3


title: Batch2-2_Tag

description:
SCT_ID10_P3 = SampleTag1
SCT_ID19_P3 = SampleTag2
SCT_ID22_P3 = SampleTag3
SCT_ID31_P3 = SampleTag4
SCT_ID35_P3 = SampleTag5
SCT_ID10_P8 = SampleTag6
SCT_ID19_P8 = SampleTag7
SCT_ID22_P8 = SampleTag8
SCT_ID31_P8 = SampleTag9
SCT_ID35_P8 = SampleTag10
The file  SampleTags.xlsx contains sample metadata

data_processing:
Data processing of scRNAseq was performed as described in Shichino et al. (Shichino et al., 2021).
Adapter trimming of sequencing data was performed by using cutadapt 3.4. Filtered reads were chunked into 16 parts for parallel processing using Seqkit 0.15.0. Filtered cell barcode reads were annotated by Python script provided by BD with minor modifications to be compatible with Python3.8. Associated cDNA reads were mapped to reference RNA (build GRCh38 release-104) using bowtie2-2.4.2 by the following parameters: -p 2 -D 20 -R 3 -N 0 -L 8 -I S,1,0.75 -norc -seed 656565 -reorder. Then, cell barcode information of each read was added to the bowtie2-mapped BAM files, and read counts of each gene in each cell barcode were counted by using mawk. Resulted count data was converted to genes x cells matrix file, and the knee-plot inflection threshold was detected using the DropletUtils package in R version 4.1.2. In addition, we further estimate background beads using the emptyDrops formula in the DropletUtils package. Cells of total read count over inflection point were considered valid cells.
For sample tag data, adapter trimming of sequencing data was performed using cutadapt 3.4 (cutadapt). Filtered reads were chunked to 64 parts for parallel processing using Seqkit 0.15.0 (Seqkit). Filtered cell barcode reads were annotated using the Python script provided by BD with a minor modification for compatible to Python3.8. Associated sample tag reads were mapped to known barcode fasta using bowtie2-2.4.2 (bowtie) with the following parameters: -p 2 -D 50 -R 20 -N 0 -L 14 -i S,1,0.75 -norc -seed 656565 -reorder -trim-to 3:40 -score-min L,-9,0 -mp 3,3 -np 3 -rdg 3,3.  The cell barcode information for each read was added to the bowtie2-mapped BAM files, and read counts of each Tag in each cell barcode were counted using mawk. The resulting count data were converted to Tags x cells matrix file using the data.table package in R 3.6.3, and the top 1M cell barcodes were extracted. For assignment of each tags to each cell barcodes, read counts of each tag in each valid cell barcode, which defined by the cDNA matrix, were extracted from tag/cell barcode expression matrix. Unassigned cell barcodes were labeled as “not-detected” cells. The sum of the total read counts of each tag was normalized to the minimum sum count of each tags, and log2 fold-change between first most tag counts and second most tag counts within each cell barcode. Each cell barcode was ranked by the fold-change ascending order, and top N cells were identified as doublets (N was calculated as theoretically detectable doublets calculated using the Poisson’s distribution based on the number of loaded cells, total Rhapsody well number, and number of tags used).
For AbSeq data, adapter trimming of sequencing data was performed using cutadapt 3.4. Filtered reads were chunked into 64 parts for parallel processing using Seqkit 0.15.0. Filtered cell barcode reads were annotated by Python script provided by BD with minor modifications to be compatible with Python3.8. Associated AbSeq reads were mapped to known barcode fasta by using bowtie2-2.4.2 by the following parameters: -p 2 -D 20 -R 3 -N 0 -L 8 -I S,1,0.75 -norc -seed 656565 -reorder-trim-to 3:39 -score-min L,-9,0 -mp 3,3 -np 3 -rdg 3,3. Then, cell barcode information of each read was added to the bowtie2-mapped BAM files, and read counts of each AbSeq tag in each cell barcode were counted using mawk. Resulted count data were converted to tags x cells matrix file by using the data.table package in R version 4.1.2, and top 1M cell barcodes were extracted.
Raw data of single-cell TCR sequencing were processed by pipeline produced by ImmunoGeneTeqs, Inc. Briefly, sequencing reads were separated by cell barcode, then adapter trimming and quality filtering of sequencing data were performed using cutadapt 3.4. PCR and sequencing errors were corrected by lighter v1.1.2 with the following parameters: -newQual 25 -maxcor 4 -K 20. Error-corrected sequence reads were processed by MiXCR55 to generate the list of TCRα or TCRβ sequences for each cell barcode, using reference human TCR V/D/J sequences registered in the international IMGT information system. Quality filtering of cell barcodes was performed with the following criteria: (1) more than 32 TCR reads were detected. (2) the proportion of the most frequent TCR sequence in cell barcodes was over 0.6. (3) the cell barcode was annotated to the T cell cluster in scRNAseq/AbSeq analysis. Parameters used in MiXCR is summarized as Supplementary Table S4.  Finally, the most frequent sequence of TCRα and TCRβ was adopted and paired in each cell barcode. Identified TCRα or TCRβ sequence was imported into the meta.data matrix in Seurat object of scRNAseq/AbSeq data based on the cellular barcode identities. T-cell clones were determined as cells with the same TCRβ sequence defined by V segment, J segment, and CDR3 nucleotide sequence. The frequency of each clone was calculated as the proportion of cells belonging to the clone in all T cells whose TCRβ sequence was assigned.
Assembly: GRCh38 release-104
Assembly: IMGT reference sequences of human TRA and TRB
Supplementary files format and content: (scRNAseq, Abseq) tab-delimited text files include raw tag-count values for each sample
Supplementary files format and content: (scTCRseq) correspondence table between cell barcodes and TCR sequences
Library strategy: single-cell CITE-seq / TCR-seq

extract_protocol:
PBMCs were isolated from whole blood with BD vacutainer CPT tubes (BD), resuspended in CELLBANKER 1 (Takara), and stored in -80°C.
PBMC thawed as described above were resuspended with Cell Staining Buffer (BioLegend) supplemented with Human TruStain FcX. After 2-3 minutes, 2x AbSeq master mix of Immune Discovery Panel (BD Biosciences) prepared as the provider’s protocol was added to the cell suspension, and stained for 15 minutes on ice. Then, the antibody mix 3 (Supplementary Table S1) with Sample Tag oligonucleotide-conjugated antibodies (Single-Cell Multiplexing Kit, BD Biosciences) was added to the cell suspension and left on ice for 15 minutes for staining. Stained cells were washed with Cell Staining Buffer.
Non-naive CD4+ and CD8+ cells (ID2 and ID27) or only non-naive CD8+ cells (ID10, ID19, ID22, ID31, and ID35) were sorted using FACS Aria II or Aria III and FACSDiva software (v8.0.1). Nonviable cells were excluded from the analysis based on forward and side scatter profiles and PI staining. Data were analyzed using FlowJo software (version 10.8.1).
Live cells were stained with Calcein AM (Nacalai) and counted using Flow-Count fluorospheres and a Cytoflex flow cytometer. About 25,000 - 30,000 labeled cells were loaded on a BD Rhapsody cartridge (BD) and processed using BD Rhapsody™ Cartridge Reagent Kit (BD) following the manufacturer’s instructions. Single-cell mRNA, AbSeq barcodes, and Sample Tag barcodes were captured by BD Rhapsody beads coated with poly(T) oligonucleotide with a unique cell barcode and molecular barcode on each bead. Single-cell cDNA synthesis was performed using BD Rhapsody cDNA Kit (BD) following the manufacturer’s protocol. Targeted mRNA, TCRseq, and Abseq Index libraries were prepared using Immune Response Panel HS (BD) according to the manufacturer’s instructions with the following modifications. (1) We used the original set of primers for TCR amplification, as summarized in Table S2. (2) We performed additional PCRs to introduce a unique dual index for sequencing (3rd PCR for mRNA and TCR), as described in Table S3. (3) We used ProNex Size-Selection DNA purification System (Promega) for the purification of PCR products, as summarized in Table S8. Amplified libraries were quantified using a KAPA SYBR Fast qPCR Kit (KAPA Biosystems), and size distribution was analyzed by Microchip Electrophoresis System MultiNA (Shimadzu). Targeted RNAseq, TCRseq, and AbSeq libraries were sequenced on an Illumina Novaseq 6000 S4 flowcell (67 bp read 1 and 140 bp read 2) using NovaSeq 6000 S4 Reagent Kit v1.5 to a depth of approximately 20,000 reads, 7,000 reads, and 10,000 reads per cell, respectively.

treatment_protocol: Thirty-eight healthcare workers (over 20 years old) at Nara Medical University in Japan received two doses of Pfizer-BNT162b2 vaccine between 10 March and 30 March 2021.

value: CD14- CD19- CD3+ [CD45RA- or CD27-] CD4- T cells

value: Multiplexed sample:ages range 34-41

value: pooled male and female

tag: condition

value: multiplexed sample:1 week after second or third shot


title: NaraCOVID_TP5_CD4_006_TCR


title: normal 1


title: KO +R837 3h [SAM24402097]

value: Bone Marrow

value: N4bp1 KO

tag: cell line

value: Bone Marrow Derived Macrophages


title: Knockout (KO), scRNAseq

first: Katherine

last: Beigel

state: PA

address: 2716 South Street

department: Department of Biomedical and Health Informatics

institute: Children's Hospital of Philadelphia

description: BAM file is from CellRanger pipeline (aligned and sorted)

data_processing:
Cell Ranger pipeline (10x Genomics, v. 3.1.0) was used to convert BCL files into FASTQ files, perform STAR alignment to mm10 genome, filter, count UMIs, and generate gene-barcode matrices.
Processed files are the barcodes.tsv, features.tsv, and matrix.mtx files from CellRanger filtered_feeature_bc_matrix output (https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/3.1/what-is-cell-ranger)
Assembly: mm10
Supplementary files format and content: Tab-separated values files and matrix files

source_name: distal colon myenteric plexus

extract_protocol:
P5 KO (Bap1 fl/fl; Tyr-Cre +; R26 TdT/TdT,  n=3 males) and WT (Bap1 wt/wt; Tyr-Cre +; R26 TdT/TdT, n=5 (3 males/ 2 females)) pups were euthanized and rapidly dissected on ice. Full-length colons were opened on the mesenteric side and the muscularis layer was removed using fine forceps. For each pup, muscularis tissue in carboxygenated (5% CO2/95% O2) 1× Hank’s balanced salt solution (HBSS; Thermo-Fisher Scientific; Cat. #14025092) was cut into small pieces with insulin needles, and dissociated with Liberase (0.625 mg/mL; Sigma-Aldrich; Cat#:5401135001) plus DNase I (0.1 mg/mL; Millipore Sigma; Cat#:11284932001), MgCl2 (6 mM; Cat#:BP214-500), in DMEM/F-12 (Thermo-Fisher Scientific; Cat#:11320033) for 40 minutes at 37°C. Cells were triturated (P1000) every 10-15 minutes during Liberase digestion. Dissociated cells were passed through Falcon 35-μm filters (Corning, Corning, NY; Cat#:352235), pelleted (150 g, 5 minutes, 4°C) after addition of 0.04% w/v BSA (Thermo-Fisher Scientific; Cat#:AM2618) in HBSS (⅓ final volume), and then resuspended in w/v 1% BSA (Thermo-Fisher Scientific; Cat#:AM2618) in HBSS. After an additional pass through Falcon 35-μm filters, the cells were sorted (BD FACSJazz, 100 μm nozzle) into 300-μL 0.04% BSA in HBSS. Sorted cells were pelleted (150 g, 5 minutes, 4°C), supernatant was removed leaving only ~30 μL and the samples were submitted for single cell RNA sequencing.
Libraries prepared with Chromium Single Cell 3′ Reagent Kits v3 (10x Genomics) according the manufacturer's instructions.

value: distal colon myenteric plexus

value: enteric neurons and glia

value: Bap1 fl/fl; Tyr-Cre +; R26 TdT/TdT

value: 5 days old (P5)

tag: group

value: 3 males

tag: strain

value: Tyr-Cre +; R26 TdT/TdT


title: KO +R837 3h [SAM24402098]

tag: treatment


title: Batch2-2_TCR

source_name: Peripheral blood T cells

tag: cell type

tag: age


title: Wild-type (WT), scRNAseq

value: Bap1 wt/wt; Tyr-Cre +; R26 TdT/TdT

value: 3 males, 2 females


title: KO +R837 3h [SAM24402099]


title: VUMC0381

source_name: plasma


title: NaraCOVID_TP5_CD4_003_TCR

value: CD4+ T cells

tag: Sex

value: male

tag: date

value: 3 month after receiving the second shot


title: Batch2-2_Target

value: Three shots of Pfizer-BNT162b2 vaccine

value: CD14- CD19- CD3+ [CD45RA- or CD27-] CD4- T cells

value: Multiplexed sample:ages range 34-41


title: NaraCOVID_TP5_CD4_001_TCR


title: WT +R837 3h +ActD 0.5h [SAM24402100]

value: Bone Marrow

value: WT


title: VUMC0382

tag: study site (plco=1 swmhs=2 sccs=3 mec=4)

tag: case control status (control=0 case=1)


title: VUMC0380

tag: sets

tag: sex (female=0 male=1)


title: VUMC0383

tag: age at diagnosis

tag: race (white=1 black=2 other=3)


title: VUMC0378


title: VUMC0379


title: Batch2-2_Abseq

value: pooled male and female

value: multiplexed sample:1 week after second or third shot


title: Spleen Th OE11

city: Oberschleißheim

first: Caspar

last: Ohnmacht

address: Ingolstädter Landstraße 1

department: Institute for Allergy Research

country: Germany

institute: Helmholtz Munich

data_processing:
Nextflow nf-Core/rnaseq
Preprocessing was performed including raw read QC, barcode extraction, trimming and removal of contaminants
Alignment and quantification using STAR via RSEM
Assembly: GRCm38
Supplementary files format and content: Gene level quantification for each sample.

source_name: Spleen

extract_protocol:
Small intestine was extracted from mice and Peyer's patches were removed. Then intestine was opened longitudinally, washed in PBS and cut into 0.5-1 cm pieces. The pieces were then incubated in ice cold PBS with 30 mM EDTA for 30 min and thoroughly washed in PBS. Afterwards they were cut into smaller pieces and digested three times for 10 min, 20 min and 30 min in RPMI 1640 containing 25 mM HEPES, 0,5 mg/ml Collagenase D and 10 µg/ml DNaseI. Disintegrated supernatants were collected in RPMI1640 + 10%FCS in between digestion steps. Cells were then filtered to remove clumps and Percoll density gradient centrifugation (40%/80%) was performed to purify lymphocytes. Spleen and lymph nodes were meshed through a 70 μm filter and then washed in DPBS. For spleens, lysis of erythrocytes was performed by incubating cells in 2 ml of ACK lysis buffer (0.15 M ammonium chloride, 10 mM potassium hydrogen carbonate, 1 mM EDTA-di sodium) for 2 min, before samples were washed and resuspended in DPBS. CD4 T cells were enriched prior to sorting using a CD4 T cell enrichment kit (Miltenyi) according to manufacturer’s protocol. Cells were stained with antibodies against CD45, CD3 and CD4 and before sorting 7AAD was added to label dead cells. They were then sorted for CD45+CD3+CD4+7AAD- and depending on Foxp3 and RORgt reporter expression using the FACS Aria III.
RNA isolation of bulk Treg populations was performed using the RNeasy Plus Micro Isolation Kit (Qiagen). Cells were sorted into lysis buffer containing β-mercaptoethanol and isolation was performed according to manufacturer's instructions.
The SMART-Seq v4 ultra-low input RNA kit (Takara) was used for cDNA synthesis using the ERCC ExFold RNA Spike-In Mixes (Invitrogen). Library preparation was done using the Illumina Nextera XT DNA Library Preparation Kit. For bead cleanup AMPure XP (Beckman Coulter) were used.

tag: tissue

value: Spleen

value: Foxp3- CD4+Tcell

tag: genotype

value: Foxp3-Cre Bcl3-OE


title: Spleen Treg OE11

value: Foxp3+ Treg


title: WT +R837 3h +ActD 0.5h [SAM24402101]

value: Bone Marrow Derived Macrophages


title: Spleen Th OE10


title: Batch2-1_TCR

tag: condition


title: WT +R837 3h +ActD 0.5h [SAM24402102]

tag: cell line


title: VUMC0377


title: KO +R837 3h +ActD 0.5h [SAM24402103]

value: N4bp1 KO


title: Spleen Treg OE10


title: VUMC0375


title: Batch2-1_Target


title: KO +R837 3h +ActD 0.5h [SAM24402104]


title: Spleen Th OE9


title: VUMC0373


title: Spleen Treg OE9


title: Batch2-1_Abseq


title: KO +R837 3h +ActD 0.5h [SAM24402105]


title: Kelly cells, shAMB, Doxycycline, replicate 3

city: Randwick

first: Murray

last: Norris

state: NSW

address: Lowy Cancer Research Centre

country: Australia

institute: Children's Cancer Institute

data_processing:
Illumina paired-end sequencing data was alligned using STAR to human genome build hg38
BAM files were sorted and indexed using SAMtools
Gene expression and isoform expression counts were calculated using RSEM
Assembly: hg38
Supplementary files format and content: tab-delimited text files containing raw and TPM counts for all samples

source_name: Cell line

extract_protocol:
Total RNA was isolated using the RNeasy Micro kit
Whole transcriptome sequencing was performed at the Ramaciotti Centre for Genomics with SMARTer Stranded Total RNA-seq v2  preparation kit. Prepared libraries were pooled and sequenced on NovaSeq 6000 S1 2x100bp lane generating an average of 60 million reads per sample.

value: Cell line

value: Kelly

value: Human Neuroblastoma

value: shAMB RUNX1T6

value: Doxycycline


title: Kelly cells, shAMB, Doxycycline, replicate 2

value: shAMB RUNX1T5


title: WT +R837 3h +ActD 1h [SAM24402106]

value: +R837 3h +ActD 1h


title: Spleen Th KO3


title: Kelly cells, shAMB, Doxycycline, replicate 1

value: shAMB RUNX1T4


title: VUMC0374


title: VUMC0376


title: Batch2-1_Tag

value: Three shots of Pfizer-BNT162b2 vaccine

value: CD14- CD19- CD3+ [CD45RA- or CD27-] CD4- T cells

value: Multiplexed sample:ages range 34-41


title: WT +R837 3h +ActD 1h [SAM24402107]

value: Bone Marrow

value: WT


title: VUMC0370


title: Spleen Treg KO3

value: Spleen


title: VUMC0372


title: VUMC0371


title: WT +R837 3h +ActD 1h [SAM24402108]


title: Kelly cells, shAMB, DMSO, replicate 3

value: shAMB RUNX1T3

value: DMSO


title: KO +R837 3h +ActD 1h [SAM24402109]

value: N4bp1 KO

value: Bone Marrow Derived Macrophages


title: Spleen Th KO2

value: Foxp3- CD4+Tcell


title: Kelly cells, shAMB, DMSO, replicate 2

value: Cell line

value: Kelly

value: Human Neuroblastoma

value: shAMB RUNX1T2


title: VUMC0369


title: VUMC0368


title: KO +R837 3h +ActD 1h [SAM24402110]

value: +R837 3h +ActD 1h


title: Spleen Th KO1


title: Kelly cells, shAMB, DMSO, replicate 1

value: shAMB RUNX1T1


title: VUMC0367


title: Spleen Treg KO1

value: Foxp3+ Treg


title: Kelly cells, sh4, Doxycycline, replicate 3

value: sh4 RUNX1T6

value: Doxycycline


title: VUMC0366


title: Kelly cells, sh4, Doxycycline, replicate 2

value: sh4 RUNX1T5


title: Spleen Th WT7

value: Wildtype


title: Spleen Th WT6

value: Spleen


title: Kelly cells, sh4, Doxycycline, replicate 1

value: sh4 RUNX1T4


title: Spleen Treg WT7


title: Kelly cells, sh4, DMSO, replicate 3

value: sh4 RUNX1T3

value: DMSO


title: Spleen Treg WT6


title: Kelly cells, sh4, DMSO, replicate 2

value: sh4 RUNX1T2


title: WT +R837 3h +ActD 2h [SAM24402113]

value: Bone Marrow

value: WT

value: Bone Marrow Derived Macrophages

value: +R837 3h +ActD 2h


title: VUMC0364


title: Spleen Treg KO2


title: WT +R837 3h +ActD 2h [SAM24402114]


title: Spleen Th WT5

value: Foxp3- CD4+Tcell


title: KO +R837 3h +ActD 2h [SAM24402115]

value: N4bp1 KO


title: KO +R837 3h +ActD 2h [SAM24402116]


title: WT +R837 3h +ActD 2h [SAM24402112]


title: VUMC0363


title: Kelly cells, sh4, DMSO, replicate 1

value: Cell line

value: Kelly

value: Human Neuroblastoma

value: sh4 RUNX1T1


title: KO +R837 3h +ActD 1h [SAM24402111]

value: +R837 3h +ActD 1h


title: VUMC0360


title: VUMC0362


title: VUMC0361


title: VUMC0359


title: Spleen Treg WT5

value: Foxp3+ Treg

value: Wildtype


title: WT +R837 3h +ActD 4h [SAM24402118]

value: +R837 3h +ActD 4h


title: Kelly cells, F1HUTG, shRNA, Doxycycline, replicate 3

value: F1HUTG (Empty Vector)

value: Doxycycline


title: Kelly cells, F1HUTG, shRNA, Doxycycline, replicate 2


title: mLN Th OE11

source_name: Mesenteric lymph nodes

value: Mesenteric lymph nodes

value: Foxp3-Cre Bcl3-OE


title: WT +R837 3h +ActD 4h [SAM24402119]

value: Bone Marrow

value: WT


title: Kelly cells, F1HUTG, shRNA, Doxycycline, replicate 1


title: KO +R837 3h +ActD 2h [SAM24402117]

value: Bone Marrow Derived Macrophages

value: +R837 3h +ActD 2h


title: WT +R837 3h +ActD 4h [SAM24402120]


title: Kelly cells, F1HUTG, shRNA, DMSO, replicate 3

value: DMSO


title: VUMC0358


title: mLN Treg OE10


title: KO +R837 3h +ActD 4h [SAM24402122]

value: N4bp1 KO


title: mLN Th OE9

value: Foxp3- CD4+Tcell


title: VUMC0357


title: Kelly cells, F1HUTG, shRNA, DMSO, replicate 2

value: Cell line

value: Kelly

value: Human Neuroblastoma


title: Kelly cells, F1HUTG, shRNA, DMSO, replicate 1


title: VUMC0355


title: KO +R837 3h +ActD 4h [SAM24402123]


title: Kelly PDX cells, RUNX1T1 KO, replicate 4

source_name: Tumour

value: Tumour

value: RUNX1T1 KO

value: Doxycycline Food


title: mLN Th KO3


title: WT +R837 7h [SAM24402124]


title: VUMC0354


title: VUMC0353


title: Kelly PDX cells, RUNX1T1 KO, replicate 3


title: VUMC0352


title: mLN Treg KO3

value: Foxp3+ Treg


title: mLN Treg OE11


title: WT +R837 7h [SAM24402125]


title: mLN Th OE10


title: Kelly PDX cells, RUNX1T1 KO, replicate 2


title: mLN Th KO2


title: Kelly PDX cells, RUNX1T1 KO, replicate 1


title: VUMC0351


title: KO +R837 3h +ActD 4h [SAM24402121]

value: +R837 3h +ActD 4h


title: VUMC0356


title: KO +R837 7h [SAM24402127]


title: VUMC0350


title: mLN Treg OE9

value: Mesenteric lymph nodes


title: KO +R837 7h [SAM24402128]


title: VUMC0349


title: Kelly PDX cells, Control, replicate 3

value: Control Food


title: mLN Th KO1


title: KO +R837 7h [SAM24402129]

value: Bone Marrow


title: mLN Treg KO1


title: KO BMDMs, R837 8h, rep 3

treatment_protocol: Time course of treatment with R837 or Pam3 for 0,2,4,6,8 hrs

tag: treatment/timepoint


title: VUMC0348


title: VUMC0347


title: mLN Th WT7

value: Wildtype


title: VUMC0346


title: Kelly PDX cells, Control, replicate 1

value: WT


title: mLN Treg KO2


title: KO BMDMs, R837 8h, rep 1

value: Bone Marrow Derived Macrophages


title: VUMC0344


title: mLN Th WT6


title: Kelly, input, Doxycycline, replicate 2

library_source: genomic

data_processing:
Illumina single-end sequencing data was aligned using bowtie2 to human genome build hg38
BAM files were sorted and indexed using SAMtools (-q 30)
ChIP peaks were identified using MACS2 (-p 1e-5)
CONT/POS Input replicate 1 was used for replicate 3 input (techincal replicate)
bigWig files were generated using deeptools (--binSize 10)
Assembly: hg38
Supplementary files format and content: bigWig, xls (MACS2 output containing peaks)

molecule: genomic DNA

source_name: Kelly

extract_protocol:
The complexes were immunoprecipitated with antibodies specific for H3K4ME1 (Active motif #39297), H3K4me2 (Active motif # 39141), H3K4ME3 (Active Motif #39159), Histone H3K27AC (Active motif #39133), and H3K27M3 (Millipore #07-449). The antibody/protein complexes were collected by Protein A/G PLUS agarose beads (Santa Cruz sc-2003) and washed several times. The immune complexes were eluted with 1% SDS and 0.1 M NaHCO3 and samples were treated with proteinase K for 1 h and DNA was purified by phenol/chloroform extraction, ethanol precipitation, and resuspended in 15 μl H2O.
In total 2.5 ng of ChIP-DNA samples were used for library preparation at the Ramaciotti Centre for Genomics with ThruPLEX DNA prep KIT. The libraries were sequenced on Illumina NovaSeq 6000 S1 single-end 100bp reads to obtain average depth of 70M reads per sample

tag: chip antibody

value: Doxycycline


title: WT +R837 7h [SAM24402126]

tag: treatment


title: Kelly, input, Doxycycline, replicate 1

tag: cell type


title: WT BMDMs, R837 8h, rep 3


title: mLN Treg WT6


title: Kelly PDX cells, Control, replicate 4


title: WT BMDMs, R837 8h, rep 2


title: Kelly, H3K4me3, Doxycycline, replicate 3

value: H3K4me3 (Active Motif #39159)


title: VUMC0343


title: Kelly PDX cells, Control, replicate 2


title: VUMC0342


title: Kelly, H3K4me3, Doxycycline, replicate 2


title: mLN Treg WT5


title: mLN Th WT5

value: Foxp3- CD4+Tcell


title: WT BMDMs, R837 8h, rep 1


title: Kelly, H3K4me3, Doxycycline, replicate 1


title: VUMC0341


title: mLN Treg WT7


title: SI tTreg KO3

source_name: Small intestine

value: Small intestine

value: RORgt- tTreg


title: KO BMDMs, R837 8h, rep 2

value: N4bp1 KO


title: KO BMDMs, R837 6h, rep 3


title: VUMC0340


title: KO BMDMs, R837 6h, rep 2


title: SI pTreg KO3

value: RORgt+ pTreg


title: Kelly, H3K4me2, Doxycycline, replicate 2

value: Kelly

value: H3K4me2 (Active motif # 39141)

value: Human Neuroblastoma


title: KO BMDMs, R837 6h, rep 1


title: Kelly, H3K4me2, Doxycycline, replicate 1


title: VUMC0338


title: VUMC0337


title: WT BMDMs, R837 6h, rep 3


title: WT BMDMs, R837 6h, rep 2


title: SI pTreg KO1


title: Kelly, H3K4me1, Doxycycline, replicate 3

value: H3K4me1 (Active motif #39297)


title: SI pTreg KO2


title: KO BMDMs, R837 4h, rep 3


title: SI tTreg KO1


title: Kelly, H3K4me1, Doxycycline, replicate 2


title: Kelly, H3K4me1, Doxycycline, replicate 1


title: WT BMDMs, R837 6h, rep 1


title: VUMC0336


title: VUMC0335


title: SI tTreg WT3


title: Kelly, H3K27me3, Doxycycline, replicate 3

value: H3K27me3 (Millipore #07-449)


title: SI pTreg WT3


title: VUMC0339


title: Kelly, H3K4me2, Doxycycline, replicate 3


title: Kelly, H3K27me3, Doxycycline, replicate 2


title: KO BMDMs, R837 4h, rep 2


title: VUMC0333


title: VUMC0334


title: KO BMDMs, R837 4h, rep 1


title: SI tTreg WT2


title: SI tTreg KO2


title: WT BMDMs, R837 4h, rep 3


title: VUMC0332


title: Kelly, H3K27me3, Doxycycline, replicate 1


title: VUMC0331


title: WT BMDMs, R837 4h, rep 2


title: VUMC0330


title: SI tTreg WT1


title: Kelly, H3K27ac, Doxycycline, replicate 2

value: H3K27ac (Active motif #39133)


title: WT BMDMs, R837 4h, rep 1

value: Bone Marrow


title: SI pTreg WT1

value: Wildtype


title: KO BMDMs, R837 2h, rep 3


title: Spleen tTreg KO3

value: Spleen


title: Kelly, H3K27ac, Doxycycline, replicate 1


title: KO BMDMs, R837 2h, rep 2

value: Bone Marrow Derived Macrophages


title: Spleen pTreg KO3


title: VUMC0329


title: Kelly, input, DMSO, replicate 2

value: DMSO


title: VUMC0328


title: Spleen tTreg KO2


title: KO BMDMs, R837 2h, rep 1


title: Kelly, input, DMSO, replicate 1


title: VUMC0327


title: WT BMDMs, R837 2h, rep 3

value: WT


title: VUMC0326


title: Kelly, H3K4me3, DMSO, replicate 3

value: H3K4me3 (Active Motif #39159)


title: SI pTreg WT2

value: Small intestine


title: VUMC0325


title: Kelly, H3K27ac, Doxycycline, replicate 3

value: Doxycycline


title: Spleen tTreg KO1

value: RORgt- tTreg


title: Kelly, H3K4me3, DMSO, replicate 2


title: WT BMDMs, R837 2h, rep 1


title: Kelly, H3K4me3, DMSO, replicate 1


title: Spleen pTreg KO1

value: RORgt+ pTreg


title: Spleen pTreg KO2


title: KO BMDMs, Pam3 8h, rep 3

value: N4bp1 KO

value: Pam3 8h


title: KO BMDMs, Pam3 8h, rep 2


title: Kelly, H3K4me2, DMSO, replicate 3

value: Kelly

value: H3K4me2 (Active motif # 39141)

value: Human Neuroblastoma


title: Kelly, H3K4me2, DMSO, replicate 2


title: VUMC0323


title: Spleen pTreg WT3


title: KO BMDMs, Pam3 8h, rep 1


title: Spleen tTreg WT3


title: Kelly, H3K4me2, DMSO, replicate 1


title: WT BMDMs, R837 2h, rep 2


title: Spleen tTreg WT2


title: VUMC0321


title: VUMC0320


title: WT BMDMs, Pam3 8h, rep 3


title: Kelly, H3K4me1, DMSO, replicate 3

value: H3K4me1 (Active motif #39297)


title: Spleen tTreg WT1


title: VUMC0319


title: Spleen pTreg WT2


title: WT BMDMs, Pam3 8h, rep 2

value: Bone Marrow


title: Kelly, H3K4me1, DMSO, replicate 2


title: Kelly, H3K4me1, DMSO, replicate 1


title: VUMC0318


title: WT BMDMs, Pam3 8h, rep 1


title: VUMC0316


title: KO BMDMs, Pam3 6h, rep 3

value: Pam3 6h


title: VUMC0322


title: Kelly, H3K27me3, DMSO, replicate 2

value: H3K27me3 (Millipore #07-449)


title: mLN pTreg KO3

value: Mesenteric lymph nodes


title: Kelly, H3K27me3, DMSO, replicate 1

value: DMSO


title: KO BMDMs, Pam3 6h, rep 2

value: Bone Marrow Derived Macrophages


title: VUMC0317


title: KO BMDMs, Pam3 6h, rep 1


title: VUMC0315


title: Kelly, H3K27ac, DMSO, replicate 3

value: H3K27ac (Active motif #39133)


title: WT BMDMs, Pam3 6h, rep 3

value: WT


title: mLN pTreg KO2


title: WT BMDMs, Pam3 6h, rep 2


title: VUMC0314


title: Kelly, H3K27me3, DMSO, replicate 3


title: Kelly, H3K27ac, DMSO, replicate 2


title: VUMC0313


title: mLN tTreg KO1


title: Spleen pTreg WT1

value: Spleen

value: Wildtype


title: VUMC0312


title: Kelly, H3K27ac, DMSO, replicate 1


title: WT BMDMs, Pam3 6h, rep 1


title: VUMC0311


title: CD274 R2

city: Boston

first: Cherubin

last: Manokaran

state: Massachusetts

address: 450 Brookline Ave

department: Cancer Biology

institute: Dana Farber Cancer Institute

data_processing:
Basecalls were performed using Illumina CASAVA 1.8
Raw reads containing adapters, base quality scores less than 5 and more than ten percent N’s were removed using Cutadapt v1.16.
Processed reads were then mapped to hg19 with STAR v2.7.3a 2-pass alignment using ENCODE parameters, first with a genome index of 100 bp overhangs and then with an index built using 75 bp overhangs and a combination of the junctions determined from the initial alignments.
Unstranded gene hits were then counted for both alignments using HTSeq v0.9.1 with a minimum alignment quality of 10, and differential expression was determined in DESeq2. As independent validation, gene-level differential expression was determined from transcript abundances quantified in Kallisto v0.46 using the hg19 transcriptome.
Assembly: hg19
Supplementary files format and content: tab-delimited text files include raw gene counts for each sample

extract_protocol:
Total RNA was purified using the Monarch Total RNA Miniprep Kit (New England Biolabs, #T2010). On-column DNase digestion was performed to remove any residual DNA. The integrity of total RNA was assessed by an Agilent Bioanalyzer 2100 at Novogene.
The cDNA library was constructed using NEBNext® Ultra™ II RNA Library Prep with Sample Purification Beads (New England Biolabs, #E7775). Briefly, a total of 400 ng RNA per sample was subjected to poly(A)-selection, followed by reverse transcription to generate cDNA. The resulting cDNA was subjected to adapter ligation and PCR amplification. Library quality control was performed using Labchip and qPCR.Sequencing was then performed on an Illumina Novaseq 6000 to generate 150 bp paired-end reads (Novogene).

value: mammary epithelial cells

value: untreated

tag: time


title: KO BMDMs, Pam3 4h, rep 3

value: N4bp1 KO

value: Pam3 4h


title: mLN pTreg KO1

value: RORgt+ pTreg


title: mLN tTreg WT3

value: RORgt- tTreg


title: mLN tTreg KO2


title: mLN tTreg KO3


title: KO BMDMs, Pam3 4h, rep 2


title: VUMC0309

data_processing: miRNAs were normalized in the following steps: 1) background correction to remove non-specific binding. The background level was calculated as an aggregate summary of seven negative control probes with no target transcript. Mean of the calculated background level for each sample was then subtracted from the adjusted probe value to remove background noise. 2) Normalization of miRNA counts using mean counts of three spike-in synthetic control miRNAs (ath-miR159a, cel-miR-248, and osa-miR414) to control for variances on the starting material and the miRNA purification efficiency. 3) Sample content normalization using geometric mean counts of the top 50 miRNAs expressed excluding hsa-miR-320e, hsa-miR-16-5p and hsa-miR-451a because these miRNAs may be related to red blood cell contamination or hemolysis. After these steps, the expression level of each miRNA was calculated.

extract_protocol: Qiagen’s miRNeasy Plasma Kit manufacturer's protocol


title: mLN pTreg WT2


title: VUMC0307


title: mLN pTreg WT3


title: KO BMDMs, Pam3 4h, rep 1


title: CD274 R1


title: EV R2


title: EV R1


title: VUMC0306


title: WT BMDMs, Pam3 4h, rep 3


title: sgPTEN Colony GSKi R2

value: sgPTEN

value: 22 hours


title: VUMC0305


title: VUMC0308


title: mLN tTreg WT1


title: WT BMDMs, Pam3 4h, rep 1

value: Bone Marrow


title: sgPTEN Colony GSKi R1


title: WT BMDMs, Pam3 4h, rep 2


title: mLN pTreg WT1


title: KO BMDMs, Pam3 2h, rep 3

value: Pam3 2h


title: VUMC0304


title: VUMC0303


title: sgPTEN Colony DMSO R1

value: vehicle (0.1 % DMSO v/v)


title: Imiquimod-treated HIF1⍺-epithelial specific KO, replicate 4

city: New York

first: Shruti

last: Naik

state: New York

address: 1 Gustave L. Levy Pl

department: Immunology & Immunotherapy

institute: Icahn School of Medicine at Mount Sinai

data_processing:
Sequencing results were demultiplexed and converted to FASTQ format using Illumina bcl2fastq software
The fastq files were trimmed for adaptor sequences using trimmomatic version 0.36 in paried end mode with parameters - minimal read length option set to 35 base pairs, trailing set to 5 and sliding window set to 4:15.
After trimming, the data was aligned using STAR. Human samples were aligned to the human genome (hg38) and mouse samples aligned to the mouse genome (mm10/GRCm38).
The featureCounts program was used to generate raw counts for each gene.
Assembly: hg38/mm10 for human/mouse respectively
Supplementary files format and content: tab-delimited text files, include raw counts for each sample. human_raw_counts contains all raw counts for human samples and mouse_raw_counts contains all raw counts for mouse samples.

source_name: IMQ skin

extract_protocol:
Total RNA was extracted using the RNeasy Fibrous Tissue Mini Kit (Qiagen) per manufacturer’s instructions.
Libraries were generated with Ribo-Zero Plus rRNA Depletion Kit (Illumina).

value: IMQ skin

value: K14 Cre; HIF1a fl/fl

value: IMQ


title: sgPTEN Colony R1


title: KO BMDMs, Pam3 2h, rep 1

first: Rohit

last: Reja

institute: Genentech

value: Bone Marrow Derived Macrophages


title: VUMC0300


title: WT BMDMs, Pam3 2h, rep 3

value: WT


title: VUMC0302


title: KO BMDMs, Pam3 2h, rep 2


title: sgPTEN Monolayer R1


title: mLN tTreg WT2

value: Mesenteric lymph nodes

value: Wildtype


title: Imiquimod-treated HIF1⍺-epithelial specific KO, replicate 3


title: VUMC0298


title: Imiquimod-treated HIF1⍺-epithelial specific KO, replicate 5


title: Imiquimod-treated HIF1⍺-epithelial specific KO, replicate 1


title: WT BMDMs, Pam3 2h, rep 1


title: sgAAVS R2

value: mammary epithelial cells

value: untreated


title: sgAAVS R1


title: VUMC0299


title: VUMC0296


title: Imiquimod-treated WT murine skin, replicate 5


title: sgPTEN Monolayer R2


title: Imiquimod-treated HIF1⍺-epithelial specific KO, replicate 2


title: Imiquimod-treated WT murine skin, replicate 4


title: WT BMDMs, Pam3 2h, rep 2


title: Imiquimod-treated WT murine skin, replicate 6


title: sgPTEN Colony R2


title: VUMC0297


title: WT BMDMs, untreated, rep 2


title: Imiquimod-treated WT murine skin, replicate 1


title: i2v2-bGH 2500 MOI 4553 donor rep 2

city: San Francisco

first: Kristen

last: Seim

address: 201 Haskins Way

institute: GraphiteBio

description:
KW-100-26
TruSeq Stranded Total RNA prep
Expression_Norm_gene (6 of the 24 samples were used for variant calling:  KW-100-13, KW-100-21, KW-100-29, KW-100-5, KW-106-13, KW-106-5)

data_processing:
CLC Genomics Workbench v 11.0.1
RNAseq reads were aligned to with STAR (v2.7.3a). Read counts for exons were computed with HTseq count using parameters outlined in the Supplemental Methods. Exon/intron counts were normalized by length such that total expression of the HBB gene (sum of wt and edited exon/intron expression) is 1 - analogous to standard TPM normalization for full length transcripts but applied to individual exons/introns.
Assembly: hg38
Supplementary files format and content: txt file w/ counts data

source_name: blood

extract_protocol:
Erythroid progenitors were snap frozen and total RNA was extracted.
Sequencing libraries were generated using an Illumina TruSeq Stranded mRNA sample prep kit and sequenced on a NovaSeq V1.5 (both Illumina, Inc.) as paired-end 2x150bp reads by SeqMatic, LLC (San Francisco, CA).

value: blood

value: Washed day 10 erythroid progenitors

value: i2v2-bGH 2500 MOI


title: KO BMDMs, untreated, rep 3

city: South San Francisco

address: 1 DNA Way

value: N4bp1 KO


title: SC17 - Periosteum and hematoma, tibia 7 days post-fracture

city: Créteil

first: Céline

last: Colnot

address: 8 rue du Général Sarrail

country: France

institute: INSERM U955

description: 10x Genomics

data_processing:
Fastq files were processed with the CellRanger pipeline with the default’s parameters
Reads were align against the mm10 reference genome
Filtered features barcodes matrices were used to perform data analysis
Assembly: mm10

source_name: Periosteum

extract_protocol:
For uninjured periosteum, tibias from 4 mice were dissected free of muscle and surrounding tissues. The epiphyses were cut and the bone marrow flushed. The periosteum was scraped from the cortex using Dissecting Chisel (10095-12, Fine Science Tools). For day 5 and 7 post-fracture, injured tibias from 4 to 9 mice were collected and the surrounded tissues were removed. The activated periosteum was scraped and collected with the hematoma. Collected tissues were minced and placed 5 min in ice-cold Nuclei Buffer (NUC101, Merck) before mechanical nuclei extraction using a glass douncer. Extraction was performed by 20 strokes of pestle A followed by 10-20 of pestle B. Nuclei suspension was filtered, centrifuged and resuspended in RNAse-free PBS (AM9624, ThermoFischer Scientific) with 2% Bovine Serum Albumin (A2153, Merck) and 0.2 U/µL RNAse inhibitor (3335399001, Roche). A second step of centrifugation was performed to reduce contamination by cytoplasmic RNAs. Sytox™ AADvanced™ (S10349, ThermoFischer Scientific) was added (1/200) to label nuclei and Sytox-AAD+ nuclei were sorted using Sony SH800.
The snRNA-seq libraries were generated using Chromium Single Cell Next GEM 3′ Library & Gel Bead Kit v.3.1 (10x Genomics) according to the manufacturer’s protocol. Briefly, 10 000 to 20 000 nuclei were loaded in the 10x Chromium Controller to generate single-nuclei gel- beads in emulsion. After reverse transcription, gel-beads in emulsion were disrupted. Barcoded complementary DNA was isolated and amplified by PCR. Following fragmentation, end repair and A-tailing, sample indexes were added during index PCR. The purified libraries were sequenced on a Novaseq (Illumina) with 28 cycles of read 1, 8 cycles of i7 index and 91 cycles of read 2. Sequencing data were processed using the Cell Ranger Count pipeline and reads were mapped on the HG38 2020-A reference human genome, with intronic and exonic sequences.

value: Periosteum


title: VUMC0290

city: Nashville

first: Cong

last: Wang

address: 2525 West End Ave

institute: Vanderbilt University


title: WT LSK 4 [RNA-seq]

city: NY

first: Tomer

last: Itkin

state: NY

address: 1300 York Avenue, A-869

department: Medicine

institute: Weill Cornell Medicine

description:
WT4
WT-4_S13_L002_R1_001
Polyadenylated mRNA
counts.txt

data_processing:
FastQC (v0.11.8) to check FASTQ quality.
Trim Galore (v0.6.4) for adapter trimming.
STAR (v2.6.1d) to align files.
Subread (v1.6.4) featureCounts to quantify gene counts.
Genome_build: mm10 (GRCm38)
Supplementary_files_format_and_content: counts.txt: Gene count matrix

source_name: LSK HSPC

growth_protocol: LSK cells were sorted and co-cultured with E4orf1 vascular niche cells in StemSpan serum free media supplemented with SCF, Flt3, and TPO and with knockout serum replacement media.

extract_protocol:
LSK HSPCs were isolated by fluorescence-activated cell sorting and subjected to RNA extraction using Arcturus PicoPure RNA isolation kit (applied Biosystems).
RNA-seq libraries of polyadenylated RNA were prepared using the TruSeq RNA Library Prep Kit v2 (Illumina) or TruSeq Stranded mRNA Library Prep Kit (Illumina) according to the manufacturer’s instructions.

treatment_protocol: Fli-1 alleles deletion was induced using 4-OHT.

value: LSK HSPC


title: VUMC0295


title: KO BMDMs, untreated, rep 2

source_name: Bone Marrow

value: Bone Marrow


title: SC12 - Periosteum and hematoma, tibia 5 days post-fracture

description: 10x Genomics


title: VUMC0289


title: i2v2-bGH 2500 MOI 4304 donor rep2

description:
KW-100-17
TruSeq Stranded Total RNA prep
Expression_Norm_gene (6 of the 24 samples were used for variant calling:  KW-100-13, KW-100-21, KW-100-29, KW-100-5, KW-106-13, KW-106-5)


title: VUMC0288


title: RNPonly 4553 donor rep3

description:
KW-106-7
TruSeq Stranded Total RNA prep
Expression_Norm_gene (6 of the 24 samples were used for variant calling:  KW-100-13, KW-100-21, KW-100-29, KW-100-5, KW-106-13, KW-106-5)

value: RNP only


title: KO BMDMs, untreated, rep 1


title: VUMC0293


title: Imiquimod-treated WT murine skin, replicate 3

value: IMQ skin


title: i2v2-bGH 2500 MOI 4304 donor rep1

description:
KW-100-1
TruSeq Stranded Total RNA prep
Expression_Norm_gene (6 of the 24 samples were used for variant calling:  KW-100-13, KW-100-21, KW-100-29, KW-100-5, KW-106-13, KW-106-5)


title: WT LSK 2 [RNA-seq]

description:
WT2
WT-2_S11_L002_R1_001
Polyadenylated mRNA
counts.txt


title: i2v2-bGH 2500 MOI 4553 donor rep3

description:
KW-106-2
TruSeq Stranded Total RNA prep
Expression_Norm_gene (6 of the 24 samples were used for variant calling:  KW-100-13, KW-100-21, KW-100-29, KW-100-5, KW-106-13, KW-106-5)


title: VUMC0292


title: WT LSK 1 [RNA-seq]

description:
WT1
WT-1_S10_L002_R1_001
Polyadenylated mRNA
counts.txt


title: VUMC0291


title: VUMC0324


title: SC22 - Periosteum and hematoma, tibia 3 days post-fracture

description: 10x Genomics


title: WT BMDMs, untreated, rep 1


title: RNPonly 4304 donor rep3

description:
KW-106-15
TruSeq Stranded Total RNA prep
Expression_Norm_gene (6 of the 24 samples were used for variant calling:  KW-100-13, KW-100-21, KW-100-29, KW-100-5, KW-106-13, KW-106-5)


title: i2v2-bGH 2500 MOI 4304 donor rep3

description:
KW-106-9
TruSeq Stranded Total RNA prep
Expression_Norm_gene (6 of the 24 samples were used for variant calling:  KW-100-13, KW-100-21, KW-100-29, KW-100-5, KW-106-13, KW-106-5)


title: i2v2-bGH 2500 MOI 4553 donor rep 1

description:
KW-100-10
TruSeq Stranded Total RNA prep
Expression_Norm_gene (6 of the 24 samples were used for variant calling:  KW-100-13, KW-100-21, KW-100-29, KW-100-5, KW-106-13, KW-106-5)


title: VUMC0285


title: WT LSK 3 [RNA-seq]

description:
WT3
WT-3_S12_L002_R1_001
Polyadenylated mRNA
counts.txt


title: WT BMDMs, untreated, rep 3


title: Imiquimod-treated WT murine skin, replicate 2


title: untreated 4553 donor rep3

description:
KW-106-8
TruSeq Stranded Total RNA prep
Expression_Norm_gene (6 of the 24 samples were used for variant calling:  KW-100-13, KW-100-21, KW-100-29, KW-100-5, KW-106-13, KW-106-5)

value: unedited


title: RNPonly 4553 donor rep2

description:
KW-100-31
TruSeq Stranded Total RNA prep
Expression_Norm_gene (6 of the 24 samples were used for variant calling:  KW-100-13, KW-100-21, KW-100-29, KW-100-5, KW-106-13, KW-106-5)


title: SC25 - Periosteum and hematoma, tibia 3 days post-fracture

description: 10x Genomics


title: MYCN Homozygote, RUNX1T1 WT, Coelic ganglia, replicate 1

data_processing:
Illumina paired-end sequencing data was alligned using STAR to mouse genome build mm10
BAM files were sorted and indexed using SAMtools
Gene expression and isoform expression counts were calculated using RSEM
Assembly: mm10
Supplementary files format and content: tab-delimited text files containing raw and TPM counts for all samples

source_name: Coelic ganglia

extract_protocol:
Total RNA was isolated using the RNeasy Micro kit
Whole transcriptome sequencing was performed at the Ramaciotti Centre for Genomics with SMARTer Stranded Total RNA-seq v2  preparation kit. Illumina NovaSeq 6000 S1 pair-end 100bp reads to obtain average depth of 100 million reads per sample.

value: Coelic ganglia

value: Neuroblastoma

value: MYCN homozygote, Runx1t1 wild-type


title: VUMC0287


title: VUMC0281


title: VUMC0286


title: WT EC 3

description:
WT3
Fli1_plus_Brain_B3_S28
Polyadenylated mRNA
counts.txt

source_name: Brain derived ECs

growth_protocol: ECs were cultured in DMEM Ham's F12 (50:50) media containing 20% fetal bovine serum, glutamine, HEPES, and antibiotics.

extract_protocol:
ECs were isolated by fluorescence-activated cell sorting and subjected to RNA extraction using Arcturus PicoPure RNA isolation kit (applied Biosystems).
RNA-seq libraries of polyadenylated RNA were prepared using the TruSeq RNA Library Prep Kit v2 (Illumina) or TruSeq Stranded mRNA Library Prep Kit (Illumina) according to the manufacturer’s instructions.

value: Brain-derived ECs

value: WT


title: SC24 - Periosteum, uninjured tibia

description: 10x Genomics


title: RNPonly 4304 donor rep1

description:
KW-100-7
TruSeq Stranded Total RNA prep
Expression_Norm_gene (6 of the 24 samples were used for variant calling:  KW-100-13, KW-100-21, KW-100-29, KW-100-5, KW-106-13, KW-106-5)


title: VUMC0279


title: KO LSK 4 [RNA-seq]

description:
KO4
KO-4_S17_L002_R1_001
Polyadenylated mRNA
counts.txt

value: Fli-1 knockout


title: SC14 - Periosteum, uninjured tibia

description: 10x Genomics


title: RNPonly 4553 donor rep1

description:
KW-100-15
TruSeq Stranded Total RNA prep
Expression_Norm_gene (6 of the 24 samples were used for variant calling:  KW-100-13, KW-100-21, KW-100-29, KW-100-5, KW-106-13, KW-106-5)


title: RNPonly 4304 donor rep2

description:
KW-100-23
TruSeq Stranded Total RNA prep
Expression_Norm_gene (6 of the 24 samples were used for variant calling:  KW-100-13, KW-100-21, KW-100-29, KW-100-5, KW-106-13, KW-106-5)


title: VUMC0284


title: VUMC0278


title: KO LSK 3 [RNA-seq]

description:
KO3
KO-3_S16_L002_R1_001
Polyadenylated mRNA
counts.txt


title: MYCN Homozygote, RUNX1T1 WT, Coelic ganglia, replicate 4


title: untreated 4304 donor rep2

description:
KW-100-24
TruSeq Stranded Total RNA prep
Expression_Norm_gene (6 of the 24 samples were used for variant calling:  KW-100-13, KW-100-21, KW-100-29, KW-100-5, KW-106-13, KW-106-5)


title: VUMC0277


title: VUMC0283


title: KO LSK 2 [RNA-seq]

description:
KO2
KO-2_S15_L002_R1_001
Polyadenylated mRNA
counts.txt


title: KO LSK 1 [RNA-seq]

description:
KO1
KO-1_S14_L002_R1_001
Polyadenylated mRNA
counts.txt


title: untreated 4553 donor rep2

description:
KW-100-32
TruSeq Stranded Total RNA prep
Expression_Norm_gene (6 of the 24 samples were used for variant calling:  KW-100-13, KW-100-21, KW-100-29, KW-100-5, KW-106-13, KW-106-5)


title: VUMC0282


title: MYCN Homozygote, RUNX1T1 WT, Tumour, replicate 1

value: Tumour


title: MYCN Homozygote, RUNX1T1 WT, Tumour, replicate 3


title: untreated 4304 donor rep1

description:
KW-100-8
TruSeq Stranded Total RNA prep
Expression_Norm_gene (6 of the 24 samples were used for variant calling:  KW-100-13, KW-100-21, KW-100-29, KW-100-5, KW-106-13, KW-106-5)

value: blood


title: untreated 4553 donor rep1

description:
KW-100-16
TruSeq Stranded Total RNA prep
Expression_Norm_gene (6 of the 24 samples were used for variant calling:  KW-100-13, KW-100-21, KW-100-29, KW-100-5, KW-106-13, KW-106-5)

value: Washed day 10 erythroid progenitors


title: MYCN Homozygote, RUNX1T1 Heterozygote, Coelic ganglia, replicate 2

value: MYCN homozygote, Runx1t1 Heterozygote


title: VUMC0276


title: VUMC0274


title: WT EC 1

description:
WT1
Fli1_plus_Brain_B1_S26
Polyadenylated mRNA
counts.txt


title: untreated 4304 donor rep3

description:
KW106-16
TruSeq Stranded Total RNA prep
Expression_Norm_gene (6 of the 24 samples were used for variant calling:  KW-100-13, KW-100-21, KW-100-29, KW-100-5, KW-106-13, KW-106-5)


title: KO EC 3

description:
KO3
Fli1-Brain_B3_S20
Polyadenylated mRNA
counts.txt


title: MYCN Homozygote, RUNX1T1 WT, Tumour, replicate 4


title: VUMC0273


title: WT EC 2

description:
WT2
Fli1_plus_Brain_B2_S27
Polyadenylated mRNA
counts.txt


title: MYCN WT, RUNX1T1 WT, Coelic ganglia, replicate 3

value: MYCN wild-type, Runx1t1 wild-type


title: KO EC 1

description:
KO1
Fli1-Brain_B1_S18
Polyadenylated mRNA
counts.txt


title: #4304 donor, HBB-SNP WX 2500 MOI, pIM-313 HDR template rep3

description:
KW-106-13
TruSeq Stranded Total RNA prep - variant calling
KW-106-13.vcf.gz
vcf file for variant calling

data_processing:
FASTQ files were used as input files to align to chromosome 11 (hg38) by minimap2 (v.2.24)
Assembly: hg38
Supplementary files format and content: csv file with variant calling

extract_protocol:
high molecular weight genomic DNA was extracted, and 5' and 3' phosphates were removed. The b-globin gene cluster was enriched by Cas9-RNPs digestion of two 10 kb fragments. PacBio SMRTbells were ligated to fragments, pooled and sequenced on a PacBio Sequel IIe
PacBio libraries were generated using the No Amp protocol


title: VUMC0275


title: KO EC 2

description:
KO2
Fli1-Brain_B2_S19
Polyadenylated mRNA
counts.txt


title: WT LSK 4 [ChIP]

data_processing:
FastQC (v0.11.8) to check FASTQ quality.
Trim Galore (v0.6.4) for adapter trimming
Bowtie2 (v2.3.5) to align reads
Picard (v2.21.1) to remove duplicate reads.
Samtools (v1.9) to remove reads multimapped (MAPQ > 30), unmapped, not primary aligned, failing platform, and mitchondrial reads.
MACS2 to call peaks
Genome_build: mm10
Supplementary_files_format_and_content: narrowPeak

growth_protocol: LSK cells were sorted and co-cultured with E4orf1 vascular niche cells in StemSpan serum free media supplemented with SCF, Flt3, and TPO and with knockout serum replacemnt media

extract_protocol:
LSK HSPCs were isolated by fluorescence-activated cell sorting and subjected to Nuclei extraction using NP40 buffer and digested into nucleosomes using Mnase.
ChIP-seq libraries of fragmented DNA were prepared using the KAPA Hyper Kit (KAPA) according to the manufacturer’s instructions.

treatment_protocol: Fli-1 alleles deletion was induced using 4-OHT

tag: organ

value: BM

value: LSK HSPCs

tag: antibody


title: #4553 donor, HBB-SNP WX 2500 MOI, pIM-313 HDR template rep3

description:
KW-106-5
TruSeq Stranded Total RNA prep - variant calling
KW-106-5.vcf.gz
vcf file for variant calling


title: MYCN Homozygote, RUNX1T1 Heterozygote, Coelic ganglia, replicate 1


title: WT_input


title: MYCN WT, RUNX1T1 WT, Coelic ganglia, replicate 4


title: MYCN Homozygote, RUNX1T1 WT, Tumour, replicate 2


title: MYCN WT, RUNX1T1 WT, Coelic ganglia, replicate 1


title: WT LSK 3 [ChIP]


title: #4304 donor, HBB-SNP WX 2500 MOI, pIM-313 HDR template rep 1

description:
KW-100-5
TruSeq Stranded Total RNA prep - variant calling
KW-100-5.vcf.gz
vcf file for variant calling

value: CD34+ cells


title: VUMC0270

tag: study site (plco=1 swmhs=2 sccs=3 mec=4)


title: VUMC0271

tag: case control status (control=0 case=1)


title: WT LSK 1 [ChIP]


title: #4304 donor, HBB-SNP WX 2500 MOI, pIM-313 HDR template rep2

description:
KW-100-21
TruSeq Stranded Total RNA prep - variant calling
KW-100-21.vcf.gz
vcf file for variant calling


title: hASC_D15_A_rep3

city: Oslo

first: Philippe

last: Collas

address: PO Box 1112 Blindern

department: Institute of Basic Medical Sciences

country: Norway

institute: University of Oslo

description:
24-21428_S17
day15_bulk_v_float.counts
adipogenesis_rpkm_tmm.tab
day15_bulk.v.float.tab
nucleolus_adipogenesis_DE.tab

data_processing:
RNA-Seqpairedreads were aligned to the hg38 genome with Hisat2(v2.1.0)using theensemblannotation(v95).
Reads ingeneswere countedusingfeaturecounts, with multimapping readsfractionally assigned using --fractionand-M.
Low abundance genes were filtered out of further analysis using edgeR's function "filterByExpr".
Normalisation between library sizes to get FPKM values was done using the trimmed mean of M-values method.
Normalisationfor differential expression was bylimma-voom(Law et al. 2014)and tests calculatedbylmFitandeBayes.
Calculations of changing expression over time were done pairwiseProlifvday0,day0 vday3 andday3 vday15;first within each donor and then combined across donors.
Assembly: hg38 ensembl95
Supplementary files format and content: Bigwig RNA-Seq coverage file generated with deeptools bamCoverage (binsize 50bp)
Supplementary files format and content: Raw read counts per gene, one column per sample
Supplementary files format and content: RPKM for each gene passing the filtering threshold in each sample.  Includes Prolif, day0, 1, 3 and 9 timepoints from previous study (GSE176020; Potolitsyna et al., 2022).
Supplementary files format and content: Differential expression analysis tables including logFC and adj.P.Val.  Analysis 1) bulk day15 versus adipocytes isolated from day15.  Analysis 2) Adipogenesis timecourse analysis with Prolif, day0 and day3 timepoints from previous study (GSE176020; Potolitsyna et al,. 2022).

source_name: Human primary adipose stem cells

growth_protocol: Human normal adipose tissue-derived mesenchymal stem cells were isolated from subcutaneous liposuction material. Cells were expanded in DMEM/F12 containing 10% fetal calf serum, and 20 ng/ml basic fibroblast growth factor (FGF2; Sigma-Aldrich F5392). Cells at passage 5-8 were used in three independent differentiations into the adipocyte lineage.

extract_protocol:
For RNA-seq, total mRNA was isolated from cells at aforementioned stages of differentiation using the Qiagen RNeasy kit and processed for Illumina library preparation and sequencing.
Illumina sequencing libraries were generated as per Illumina protocols.

treatment_protocol: For adipogenic differentiation, confluent cells were cultivated for 48h in DMEM/F12/10% fetal bovine serum without growth factors. Adipogenesis was then induced by addition of 10 µg/ml insulin, 200 µM indomethacin, 0.5 µM 1-methyl-3 isobutylxanthine and 1 µM dexamethasone. Medium was renewed every 3 days until day 9. Cells were then maintained in DMEM/F12/10% fetal bovine serum and 10 µg/ml insulin until day 15. RNA from the total cell population ("bulk") was harvested by direct lysis in the well. For mature adipocyte isolation, D15 samples were trypsinized, resuspended in HBSS, 0,5% BSA, and centrifuged 200g for 5 min. Floating mature adipocytes were harvested in Trizol and processed for RNA extraction.

value: Human primary adipose stem cells

tag: time point

tag: adipose depot

value: Abdominal subcutaneous

tag: culture

value: Cultured in vitro

tag: cell fraction

value: bulk


title: VUMC0272

tag: sets

tag: sex (female=0 male=1)

tag: age at diagnosis

tag: race (white=1 black=2 other=3)


title: MYCN WT, RUNX1T1 WT, Coelic ganglia, replicate 2

tag: tissue

value: Coelic ganglia

value: Neuroblastoma

tag: genotype


title: #4553 donor, HBB-SNP WX 2500 MOI, pIM-313 HDR template rep1

description:
KW-100-13
TruSeq Stranded Total RNA prep - variant calling
KW-100-13.vcf.gz
vcf file for variant calling

tag: treatment


title: KO LSK 4 [ChIP]

tag: cell type


title: VUMC0269


title: VUMC0267


title: MYCN Homozygote, RUNX1T1 WT, Coelic ganglia, replicate 3

value: MYCN homozygote, Runx1t1 wild-type


title: KO_input


title: WT LSK 2 [ChIP]

value: WT


title: #4553 donor, HBB-SNP WX 2500 MOI, pIM-313 HDR template rep2

description:
KW-100-29
TruSeq Stranded Total RNA prep - variant calling
KW-100-29.vcf.gz
vcf file for variant calling


title: hASC_D15_G_rep3

description:
21-21425_S54
day15_bulk_v_float.counts
adipogenesis_rpkm_tmm.tab
day15_bulk.v.float.tab
nucleolus_adipogenesis_DE.tab

value: Gluteal


title: hASC_D15_A_rep2

description:
23-21427_S10
day15_bulk_v_float.counts
adipogenesis_rpkm_tmm.tab
day15_bulk.v.float.tab
nucleolus_adipogenesis_DE.tab


title: VUMC0268


title: SV481_CO_KO_CT17

city: Freising

first: Yunhui

last: Niu

address: Gregor-Mendel-Str. 2

institute: Technische Universität München

data_processing:
The quality of Next Generation Sequencing data was assessed with FastQC v0.11.5
Adapter content and low quality reads were removed using Trimmomatic v0.39 (Anthony et al., 2014)
Trimmed FASTQ files were then mapped against the mouse mm10 genome with the STAR v2.7.5c aligner
Format conversions were performed using samtools v1.3.1 (Li et al., 2009). The featureCounts program v1.4.6 (Liao et al., 2014) was used to count reads located within an exon, do not overlap multiple features, with a threshold of MAPQ > = 4 and are not chimeric.
DESeq2 version 1.22.0 (RRID:SCR_015687 (Love et al., 2014) was used to normalize the read count matrix and perform differential expression analysis.
Assembly: mm10
Supplementary files format and content: txt file includes feature counts for all samples in one table

source_name: Colon

extract_protocol:
RNA was harvested using NucleoSpin® RNA kit (MACHEREY-NAGEL).
RNA libraries for RNA-seq were prepared using TruSeq Stranded mRNA Library Prep Kit following manufacturer's protocols.

value: Colon

tag: time


title: VUMC0266


title: hASC_D15_G_rep2

description:
20-21424_S47
day15_bulk_v_float.counts
adipogenesis_rpkm_tmm.tab
day15_bulk.v.float.tab
nucleolus_adipogenesis_DE.tab


title: MYCN WT, RUNX1T1 Heterozygote, Coelic ganglia, replicate 4

value: MYCN wild-type, Runx1t1 Heterozygote


title: SV469_CO_WT_CT1


title: KO LSK 1 [ChIP]

tag: organ

value: Fli-1 knockout


title: KO LSK 2 [ChIP]

tag: antibody


title: MYCN WT, RUNX1T1 Heterozygote, Coelic ganglia, replicate 3


title: VUMC0264

tag: study site (plco=1 swmhs=2 sccs=3 mec=4)


title: SV447_CO_WT_CT5


title: SV418_CO_KO_CT9


title: VUMC0263

tag: case control status (control=0 case=1)


title: hASC_D15_G_rep1

description:
19-21423_S40
day15_bulk_v_float.counts
adipogenesis_rpkm_tmm.tab
day15_bulk.v.float.tab
nucleolus_adipogenesis_DE.tab

tag: time point