title: LNCaP KD4OE-3 (C3)

first: Danthasinghe

middle: Badrajee

last: Piyarathna

state: United States

address: One Baylor Plaza

institute: Baylor College of Medicine

data_processing:
Fastq files were run through fastqc for primary qc
Sequence reads were trimmed for adaptor sequence/low-quality sequence using cutadapt (parameter- Default settings)
Trimmed sequence reads were mapped to hg19 using HISAT2
Read count extraction and normalization were performed using htseq-counts
Assembly: hg19
Supplementary files format and content: tab-delimited text files include htseq counts

source_name: LNCaP

growth_protocol: LNCaP cells were maintained in RPMI supplemented with 10% fetal bovine serum (FBS) and 1% penicillin streptomycin in humidified atmosphere with 5% CO2 at 37C

extract_protocol:
RNA was harvested using Rneasy mini plus kit (Qiagen). 1 ug of total RNA was used for the construction of sequencing libraries.
RNA libraries for RNA-seq were prepared using NovaSeq-PE-150 .

treatment_protocol: 3ug/mL Puromycin and 1 mg/mL G418 were used as respective selection antibiotics for knockdown and rescue respectively

value: LNCaP

value: Prostate epithelial cancer cell

value: UGT2B28 rescue

tag: time


title: fibroblast cells of IRF9 patient stimulated with IFNa for 8h

status: Public on May 12 2023

type: SRA

city: Paris

first: Jeremy

last: Manry

address: 24 boulevard du Montparnasse

country: France

institute: INSERM, Institut Imagine

library_source: transcriptomic

data_processing:
The RNA-seq fastq files were first inspected with FastQC (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/) to ensure that the raw data were of high quality.
For each subject, the two fastq files generated were then mapped to the human reference genome (Ensembl GRCh37 release 75) with STAR v.2.7.3a in the two-pass mode
The mapping quality of each bam file was then evaluated with RSeQC
Reads were quantified to generate gene-level feature counts from the read mapping, with HTSeq-count v.0.11.2
Assembly: Ensembl GRCh37 release 75
Supplementary files format and content: Raw counts were obtained using HTSeq-count v.0.11.2

molecule: total RNA

organism: Homo sapiens

source_name: primary fibroblasts

extract_protocol:
Total RNA was extracted from primary fibroblasts from patients or healthy controls with the the miRNeasy Mini Kit (Zimo), and treated with DNAse (Zymo) to remove residual genomic DNA. Quality controls were performed and all RNA samples were of good quality (RIN>8).
RNA-Seq libraries were prepared with the Illumina RiboZero TruSeq Stranded Total RNA Library Prep Kit (Illumina) and sequenced on the Illumina NextSeq platform in the 150-nt (for monocyte-derived and iPSC-derived macrophages) or 100-nt (for primary fibroblasts), paired-end configuration.

tag: cell type

value: primary fibroblasts

tag: disease state

value: IRF9 patient

tag: stimulation

value: IFNa

tag: treatment dose

tag: stimulation duration

value: 8 hours


title: fibroblast cells of IRF9 patient stimulated with IFNa for 2h

value: 2 hours


title: CD8_NoQ_4h_A

city: Nashville

first: Jeff

last: Rathmell

state: TN

address: 1211 Medical Center Drive

department: PMI

country: USA

institute: Vanderbilt University Medical Center

data_processing:
library quality were evaluated by FastQC
alignment were done with bowtie2 or STAR
CUT&Run peaks were called using MACS2 and IDR
RNAseq qunatification were done using featureCounts
heatmaps were generated by deeptools
Assembly: mm10
Supplementary files format and content: txt file containing peaks
Supplementary files format and content: txt file containing raw counts for RNAseq

organism: Mus musculus

source_name: CD8+ T cell, Glutamine free, 4h, RNAseq

growth_protocol: OT-I mouse splenocytes were ACK-lysed and resuspended to 2 million splenocytes/mL in complete RPMI media supplemented with 10% FBS, pen/strep, HEPES, glutamine, and β-mercaptoethanol, or in complete RPMI without glutamine (No Q).

extract_protocol:
For CUT&RUN, genomic DNA was release by Mnase. For RNAseq, RNA was extracted use Quick-RNA™ Microprep Kit.
For CUT&RUN, libraries were preparaed by KAPA HpyerPrep Kit. For RNAseq, stranded library preparations were used

treatment_protocol: OT-I cells were activated with 100 IU/mL recombinant human IL-2 (rhIL-2, NCI) and 10ng/mL SIINFEKL peptide (Sigma-Aldrich S7951). CB-839 (MedChemExpress HY-12248) or equal volume DMSO vehicle and DON (Sigma-Aldrich D2131) or equal volume PBS vehicle were added to a final concentration of 1μM (14, 17, 19). 2-deoxyglucose (Cayman Chemical 14325) or equal volume water vehicle was added to a final concentration of 1mM.

tag: cell line

value: OT-I

value: CD8+ T cell

tag: treatment

value: Glutamin Free


title: LNCaP KD4OE-2 (C2)

city: Houston

tag: genotype


title: fibroblast cells of IRF9 patient stimulated with IFNa for 30min

value: 30 minutes


title: CD8_DON_4h_D

source_name: CD8+ T cell, DON, 4h, RNAseq

value: DON


title: Cas9_nogRNA_vivo_12h_In

city: GuangZhou

first: Yan

last: Li

state: GuangDong

address: No. 135, Xingang Xi Road, Guangzhou, 510275, P. R. China

department: School of Life Science

country: China

institute: Sun Yat-sen University

library_source: genomic

data_processing:
Quality of all reads was inspected using FastQC. Low-quality and adapter-containing reads were trimmed from raw data using Cutadapt. Then, the filtered reads were mapped to the reference human genome (hg19) using the bowtie2 aligner with default settings. The SAM files were subsequently converted to the BAM file format, sorted and indexed using SAMtools. Duplicate reads were removed using picard-tools.
Peaks were called using MACS (version 2.1.2) with default settings (-p 0.01). No-sgRNA (CRISPR alone) and GFP samples were used as negative control. Common peaks between the edited and control sample were removed by BEDtools intersect command. We then compared the results with ENCODE DAC Exclusion List Regions (ENCODE #: ENCSR636HFF) to filter out artifacts using BEDtools intersect command. The rest of peaks in edited sample were candidates of off-target sites.
CROss-seq sequencing data was analyzed using custom computational pipeline. The scoring program used for capturing off-target cleavage sites or editing sites have illustrated in paper.
Genome_build: hg19
Supplementary_files_format_and_content: XLS files include peaks for each sample, TDF files for loaded in IGV for visualization

molecule: genomic DNA

source_name: HEK293T cells

growth_protocol: HEK293T cells were cultured in Dulbecco's modified eagle medium supplemented 10% fetal bovine serum and 1% penicillin/streptomycin.

extract_protocol:
Genomic DNA was freshly extracted using FastPure Cell/Tissue DNA Isolation MiniKit and stored in 25 mM K3BO3 buffer (pH 7.0) at -20 °C. Treated genomic DNA was  purified by DNeasy Blood & Tissue Kit and eluted by  25 mM K3BO3 buffer (pH 7.0).
For library preparation, fragmented DNA first be repaired and ligated to adapters, then, the IP sample was subjected to biotin enrichment using Dynabeads MyOne Streptavidin C1 (Thermo, 65001). All reactions were followed the protocol of VAHTS Universal DNA Library Prep Kit for Illumina V3 (Vazyme, ND607) with some modifications. The biotin pull-down experiments were performed following the recommendations from the Dynabeads MyOne Streptavidin C1 (Thermo, 65001) with some modifications.
CROss-seq (biotin enriched ChIP-seq), Amplicon

treatment_protocol: CROss-seq in vivo: 6-well-plate HEK293T cells were transfected with px330, ABE8e, pCMV-BE4max, and pUC19-Cas9 sgRNA expression plasmids (3 μg/well) using PEI. To perform N3-kethoxal labeling, 24 hours after transfection, previous medium was removed and pre-warmed (37 ˚C) cell culture medium with 5mM N3-kethoxal was used to incubate cells for 45min at 37 ˚C. Genomic DNA was then extracted by FastPure Cell/Tissue DNA Isolation MiniKit and eluted by 25 mM K3BO3 solution (pH 7.0). CROss-seq in vitro: 8 μg Genomic DNA extracted from HEK293T cells was incubated with 300 nM recombinant proteins (SpCas9, ABE7.10, or BE3), 900 nM sgRNA, 0.05mM N3-kethoxal, and 80 μL 5 × CRB buffer (50 mM MgCl2, 750 mM KCl, 100 mM HEPES (pH=7.4), 500 mM Sodium cacodylate) in a 400 μL reaction mix for 2 h at 37 °C. Treated genomic DNA was then purified by DNeasy Blood & Tissue Kit and eluted by using 50 μL 25 mM K3BO3 (pH 7.0).

value: embryonic kidney cells

tag: tissue source

value: kidney

tag: sample type

value: input

tag: guide rna

value: no

tag: vivo or vitro

value: in vivo

tag: editor


title: Cas9_nogRNA_vivo_12h_IP

value: IP


title: LNCaP KD4OE-1 (C1)

value: LNCaP

value: Prostate epithelial cancer cell

value: UGT2B28 rescue


title: fibroblast cells of IRF9 patient not stimulated

value: primary fibroblasts

value: IRF9 patient

value: no treatment


title: Cas9_HBG_vivo_12h_IP

value: HBG-sgRNA


title: CD8_DON_4h_C

value: OT-I

value: CD8+ T cell


title: LNCaP KD4-3 (B3)

value: UGT2B28 knockdown


title: fibroblast cells of IFNAR1 patient stimulated with IFNg for 8h

value: IFNAR1 patient

value: IFNg

value: 8 hours


title: Cas9_HBG_vivo_12h_In

value: embryonic kidney cells

value: kidney

value: input


title: CD8_DON_4h_B

value: DON


title: LNCaP KD4-2 (B2)

value: LNCaP


title: fibroblast cells of IFNAR1 patient stimulated with IFNg for 2h

value: 2 hours


title: fibroblast cells of IFNAR1 patient stimulated with IFNg for 30min

value: 30 minutes


title: LNCaP KD4-1 (B1)

value: Prostate epithelial cancer cell


title: CD8_CB839_4h_D

source_name: CD8+ T cell, CB839, 4h, RNAseq


title: fibroblast cells of IFNAR1 patient stimulated with IFNa for 8h

value: primary fibroblasts

value: IFNa


title: LNCaP NT-3 (A3)

value: Control


title: CD8_DON_4h_A

value: OT-I


title: CD8_CB839_4h_C

value: CD8+ T cell


title: Cas9_HBG_vivo_24h_In

value: HBG-sgRNA

value: in vivo


title: Cas9_HBG_vivo_24h_IP

value: IP


title: fibroblast cells of IFNAR1 patient stimulated with IFNa for 2h

value: IFNAR1 patient


title: LNCaP NT-2 (A2)


title: fibroblast cells of IFNAR1 patient stimulated with IFNa for 30min


title: LNCaP NT-1 (A1)


title: Cas9_GFP_vivo_24h_IP

value: embryonic kidney cells

value: GFP


title: CD8_CB839_4h_B


title: CD8_CTL_4h_D

source_name: CD8+ T cell, Vehicle, 4h, RNAseq

value: Vehicle


title: RNA_PostPVMF_Patient1

city: New York

first: Richard

last: Koche

state: New York

institute: Memorial Sloan Kettering Cancer Center

data_processing:
RNA-seq reads were filtered for quality and 3' trimmed for adapter sequences.
Reads were aligned to mouse genome assembly hg19 using STAR (v2.4) with default parameters.
Read count tables were created using HTSeq v0.6.1.
Normalization and differential expression analysis was performed with DESeq2.
Genome_build: hg19
Supplementary_files_format_and_content: Tab-delimited text file with un-normalized read counts over all genes.

extract_protocol:
RNA was isolated by TRIzol extraction
Libraries were prepared according to Illumina's instructions accompanying the TruSeq RNA Sample Preparation Kit

value: CD34+ peripheral blood mononuclear cells (PBMCs)

value: myeloproliferative neoplasm (MPN)

tag: il8 status

value: IL8 high


title: RNA_PVtoMF_Patient9

source_name: peripheral blood mononuclear cells (PBMCs)

value: IL8 low


title: CD8_CB839_4h_A


title: Cas9_GFP_vivo_24h_In

value: kidney

value: input


title: RNA_PV_Patient13


title: CD8_CTL_4h_C


title: Cas9_HBG_vitro_In

value: in vitro


title: Cas9_nogRNA_vivo_24h_In

value: no


title: fibroblast cells of IFNGR2 patient stimulated with IFNg for 2h

value: primary fibroblasts

value: IFNGR2 patient

value: IFNg

value: 2 hours


title: Cas9_nogRNA_vivo_24h_IP

value: IP

value: in vivo


title: fibroblast cells of IFNGR2 patient stimulated with IFNg for 8h

value: 8 hours


title: CD8_CTL_4h_B

value: OT-I

value: CD8+ T cell


title: Cas9_HBG_vitro_5mM_IP

value: embryonic kidney cells

value: HBG-sgRNA


title: RNA_PMF_Patient8

value: CD34+ peripheral blood mononuclear cells (PBMCs)

value: myeloproliferative neoplasm (MPN)

value: IL8 high


title: fibroblast cells of IFNGR2 patient stimulated with IFNg for 30min

value: 30 minutes


title: fibroblast cells of IFNGR2 patient stimulated with IFNa for 8h

value: IFNa


title: RNA_PMF_Patient2


title: Cas9_HBG_vitro_0mM_IP

value: kidney

value: in vitro


title: CD8_CTL_4h_A

value: Vehicle


title: Cas9_HBG_vitro_0.5mM_IP


title: fibroblast cells of IFNAR1 patient not stimulated

value: primary fibroblasts

value: IFNAR1 patient

value: no treatment


title: RNA_ET_Patient15

value: IL8 low


title: CD8_unstimulated_C

source_name: CD8+ T cell, unstimulated, RNAseq


title: CD8_unstimulated_D


title: Cas9_HBG_vitro_0.05mM_IP

value: IP


title: fibroblast cells of IFNGR2 patient stimulated with IFNa for 2h

value: IFNGR2 patient

value: 2 hours


title: RNA_ETtoMF_Patient10


title: Cas9_HBG_vitro_0.005mM_IP

value: embryonic kidney cells


title: CD8_unstimulated_B

value: OT-I

value: CD8+ T cell


title: fibroblast cells of IFNGR2 patient stimulated with IFNa for 30min


title: fibroblast cells of IFNGR2 patient not stimulated


title: RNA_ET_Patient14

value: CD34+ peripheral blood mononuclear cells (PBMCs)

value: myeloproliferative neoplasm (MPN)


title: CD8_unstimulated_A


title: fibroblast cells of IFNGR1 patient stimulated with IFNg for 8h

value: IFNGR1 patient

value: IFNg

value: 8 hours


title: ATAC_PostPVMF_Patient1

data_processing:
ATAC-seq reads were filtered for quality and 3' trimmed for adapter sequences.
Reads were aligned to mouse assembly hg19 using bowtie2 (v2.2.2) with default parameters.
To create density files, read overlap density was calculated with an extension of 0 bp, the signal was then normalized to 10 million mapped fragments and converted to BigWig using BEDTools.
Genome_build: hg19
Supplementary_files_format_and_content: BigWig files containing read densities normalized to 10 million mapped reads

extract_protocol:
Cells were lysed and incubated with transposition reaction mix containing PBS, 1% Digitonin, Tween-20, and Transposase (llumina). Samples were incubated for 30 minutes at 37C in a thermomixer at 1000rpm. Prior to amplification, samples were concentrated with the DNA Clean and Concentrator Kit-5 (Zymo).
Samples were eluted in 20uL of elution buffer and PCR-amplified using the NEBNext 2X Master Mix (NEB) for 10 cycles and sequenced on a HiSeq 2500 (Illumina).

value: IL8 high


title: KO Input ChIP-seq

first: Jiexi

state: TX

address: 1881 East Road

institute: The University of Texas MD Anderson Cancer Center

data_processing:
NextSeq RTA (version 2.4.11.0) was used for base calling
bcl2fastq (version 2.20.0) was used to convert the .bcl files into .fastq files
Fastq files were aligned to the mouse reference genome (mm9) using Bowtie (version 1.2.2)
SAMtools (version 1.9) was used to convert .sam file to .bam file
Deeptools (version 2.7.15) was used to generate .bigwig files
Peaks were called using MACS (version 1.4.2)
Assembly: mm9
Supplementary files format and content: bigwig

source_name: KO

growth_protocol: Cells were cultured in DMEM with 10% tetracycline-free Fetal Bovine Serum (FBS), 1X penicillin/streptomycin (Invitrogen) and 1 µg/ml doxycycline.

extract_protocol:
iKAP cells were harvested via cross-linking with formaldehyde followed by quenching with glycine. Then cells were washed with ice-cold PBS and lysed on ice for 30 minutes in ChIP harvest buffer. Lysed cells were sonicated with a Bioruptor (Diagenode) to obtain chromatin fragments (~200-500bp) and soluble chromatin fractions were obtained by centrifugation. Ten percent of supernatant was set aside as input. Soluble chromatin was diluted five times using ChIP dilution buffer with Mini Protease Inhibitor Cocktail (Roche). Anti-H3K4me1 (Cell Signaling Technology, Cat#5326, 1:50), anti-H3K4me2 (Abcam, Cat#ab7766, 5 µg for 3 x 106 cells), anti-H3K4me3 (Abcam, Cat#ab8580, 5 µg for 3 x 106 cells), anti-H3K27ac (Abcam, Cat#ab4729, 5 µg for 3 x 106 cells), anti-mouse STAT4 (Invitrogen, Cat#714500, 1:100) antibodies and Dynabeads Protein G (Invitrogen) were incubated at 4 °C followed by washing with ChIP dilution buffer. Antibodies and beads were added to samples and incubated with rotation at 4 °C overnight. On the next day, magnetic protein G beads-antibody-protein complexes were separated by placing the sample tube on a magnetic rack. The samples were then washed in RIPA wash buffer, RIPA-500 wash buffer and LiCl wash buffer. Together with input, samples were reverse crosslinked with direct elution buffer, RNase A (Invitrogen) and Proteinase K (Invitrogen) overnight. On the next day, the supernatant was transferred to a new tube and 2.3X AMPure XP beads (Beckman Coulter) were added to purify the DNA. DNA was washed with 70% ethanol and eluded with 10 mM Tris-Cl buffer.
The ChIP-seq library was prepared using KAPA HyperPrep Kit (Roche) following manufacturer’s protocol

treatment_protocol: Doxycycline was removed for 24hr.

value: KO

value: mouse CRC cell line

tag: chip antibody

value: input


title: L2-3 Input ChIP-seq 2


title: CD8_NoQ_48h_H3K27me3_B

data_processing:
library quality were evaluated by FastQC
alignment were done with bowtie2 or STAR
CUT&Run peaks were called using MACS2 and IDR
RNAseq qunatification were done using featureCounts
heatmaps were generated by deeptools
Assembly: mm10
Supplementary files format and content: txt file containing peaks
Supplementary files format and content: txt file containing raw counts for RNAseq
Library strategy: CUT&RUN

source_name: CD8+ T cell, Glutamine free, 48h, H3K27me3

value: Glutamin Free


title: fibroblast cells of IFNGR1 patient stimulated with IFNg for 2h

value: primary fibroblasts


title: 3346 Input ChIP-seq


title: ATAC_PV_Patient13

value: IL8 low


title: CD8_NoQ_48h_H3K27me3_A


title: ATAC_PMF_Patient2


title: KO H3K27ac ChIP-seq

value: anti-H3K27ac (Abcam, Cat#ab4729)


title: ATAC_MF_Patient8


title: CD8_DON_48h_H3K27me3_B

source_name: CD8+ T cell, DON, 48h, H3K27me3

value: DON


title: L2-3 H3K27ac ChIP-seq


title: fibroblast cells of IFNGR1 patient stimulated with IFNa for 8h

value: IFNa


title: fibroblast cells of IFNGR1 patient stimulated with IFNa for 2h

value: 2 hours


title: ATAC_ET_Patient14


title: 3346 H3K27ac ChIP-seq


title: fibroblast cells of IFNGR1 patient stimulated with IFNa for 30min

value: 30 minutes


title: fibroblast cells of IFNGR1 patient not stimulated

value: no treatment


title: CD8_CB839_48h_H3K27me3_A

source_name: CD8+ T cell, CB839, 48h, H3K27me3

value: OT-I

value: CD8+ T cell


title: KO H3K4me3 ChIP-seq

value: anti-H3K4me3 (Abcam, Cat#ab8580)


title: L2-3 H3K4me3 ChIP-seq


title: Osteosarcoma from p53R172H/fl group 865

first: Xiaoping

last: Su

state: Texas

address: 7300 Brompton St, Apt 5633

institute: MD Anderson Cancer Center

description: Osteosarcoma from p53R172H/fl group

data_processing:
Illumina Casava1.7 software used for basecalling.
Raw data FASTQ files were generated by standard illumina base calling pipeline. Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence, Reads were aligned to GRCm38 reference genome using STAR with default parameters
STAR was used to generate the "raw count" from an aligned bam againt transcript file
Assembly: GRCm38
Supplementary files format and content: tab-delimited text file includes raw counts for each annotated gene for each sample

source_name: Primary osteosarcoma

extract_protocol:
Total RNA was isolated from primary murine osteosarcomas that were flash frozen upon extraction, then crushed for RNA extraction using Zymo Direct-Zol RNA Extraction kit by standard protocols
RNA libraries were prepared for sequencing using standard Illumina protocols
Stranded mRNA-Seq

value: Primary osteosarcoma


title: Osteosarcoma from p53R172H/fl group 847

description: Osteosarcoma from p53R172H/fl group


title: Osteosarcoma from p53R172H/fl group 5798

description: Osteosarcoma from p53R172H/fl group


title: fibroblast cells of IFNGR1 patient stimulated with IFNg for 30min

value: IFNGR1 patient

value: IFNg


title: fibroblast cells of P2 patient stimulated with IFNg for 8h

value: IRF1 patient

value: 8 hours


title: Osteosarcoma from p53R172H/fl group 383

description: Osteosarcoma from p53R172H/fl group


title: 3346 H3K4me3 ChIP-seq

value: mouse CRC cell line


title: L2-3 H3K4me2 ChIP-seq

value: anti-H3K4me2 (Abcam, Cat#ab7766)


title: KO H3K4me2 ChIP-seq

value: KO


title: fibroblast cells of P2 patient stimulated with IFNg for 30min

value: primary fibroblasts


title: Osteosarcoma from p53R172H/fl group 627

description: Osteosarcoma from p53R172H/fl group


title: CD8_NoQ_48h_H3K27ac_B

source_name: CD8+ T cell, Glutamine free, 48h, H3K27ac

value: Glutamin Free


title: fibroblast cells of P2 patient stimulated with IFNg for 2h


title: CD8_CTL_48h_H3K27me3_A

source_name: CD8+ T cell, Vehicle, 48h, H3K27me3

value: Vehicle


title: fibroblast cells of P2 patient stimulated with IFNa for 8h

value: IFNa


title: CD8_NoQ_48h_H3K27ac_A


title: CD8_CB839_48h_H3K27me3_B


title: CD8_DON_48h_H3K27me3_A

value: DON


title: CD8_DON_48h_H3K27ac_B

source_name: CD8+ T cell, DON, 48h, H3K27ac


title: Osteosarcoma from p53R172H/fl group 622

description: Osteosarcoma from p53R172H/fl group


title: 3346 H3K4me2 ChIP-seq


title: Osteosarcoma from p53R172H/fl group 621

description: Osteosarcoma from p53R172H/fl group


title: fibroblast cells of P2 patient stimulated with IFNa for 2h

value: 2 hours


title: KO H3K4me1 ChIP-seq

value: anti-H3K4me1 (Cell Signaling Technology, Cat#5326)


title: CD8_CTL_48h_H3K27me3_B

value: OT-I

value: CD8+ T cell


title: CD8_DON_48h_H3K27ac_A


title: Osteosarcoma from p53R172H/fl group 1383

description: Osteosarcoma from p53R172H/fl group

value: Primary osteosarcoma


title: fibroblast cells of P2 patient stimulated with IFNa for 30min

value: 30 minutes


title: L2-3 H3K4me1 ChIP-seq


title: CD8_CB839_48h_H3K27ac_B

source_name: CD8+ T cell, CB839, 48h, H3K27ac


title: fibroblast cells of P2 patient not stimulated

source_name: primary fibroblasts

value: no treatment


title: CD8_CB839_48h_H3K27ac_A


title: 3346 H3K4me1 ChIP-seq


title: Osteosarcoma from p53R245W/fl group 1817

description: Osteosarcoma from p53R245W/fl group


title: Osteosarcoma from p53R245W/fl group 1580

description: Osteosarcoma from p53R245W/fl group


title: fibroblast cells of P1 patient stimulated with IFNg for 8h

value: IRF1 patient

value: IFNg


title: L2-3 Input ChIP-seq

value: mouse CRC cell line

value: input


title: Osteosarcoma from p53R172H/fl group 1441

description: Osteosarcoma from p53R172H/fl group


title: CD8_CTL_48h_H3K27ac_A

source_name: CD8+ T cell, Vehicle, 48h, H3K27ac


title: fibroblast cells of P1 patient stimulated with IFNg for 2h


title: Osteosarcoma from p53R172H/fl group 1241

description: Osteosarcoma from p53R172H/fl group


title: fibroblast cells of P1 patient stimulated with IFNg for 30min


title: CD8_CTL_48h_H3K27ac_B


title: 3346 Dox off 24hr Input ChIP-seq


title: L2-3 STAT4 ChIP-seq

value: anti-mouse STAT4 (Invitrogen, Cat#714500)


title: Osteosarcoma from p53R172H/fl group 472

description: Osteosarcoma from p53R172H/fl group


title: Osteosarcoma from p53R172H/fl group 1439

description: Osteosarcoma from p53R172H/fl group


title: fibroblast cells of P1 patient stimulated with IFNa for 8h

value: primary fibroblasts

value: 8 hours


title: 3346 Dox on Input ChIP-seq


title: Osteosarcoma from p53R172H/fl group 1509

description: Osteosarcoma from p53R172H/fl group


title: fibroblast cells of P1 patient stimulated with IFNa for 2h


title: Osteosarcoma from p53R172H/fl group 476

description: Osteosarcoma from p53R172H/fl group


title: Osteosarcoma from p53R172H/fl group 889

description: Osteosarcoma from p53R172H/fl group


title: 3346 Dox off 24hr STAT4 ChIP-seq


title: fibroblast cells of P1 patient stimulated with IFNa for 30min


title: 3346 Dox on STAT4 ChIP-seq


title: fibroblast cells of P1 patient not stimulated


title: Osteosarcoma from p53R172H/fl group 524

description: Osteosarcoma from p53R172H/fl group


title: Osteosarcoma from p53fl/fl group 4183

description: Osteosarcoma from p53fl/fl group


title: Osteosarcoma from p53fl/fl group 475

description: Osteosarcoma from p53fl/fl group


title: Murine osteosarcoma cell line expressing p53R245W/- Gla_941_R3

data_processing:
Basecalls were performed using bcl2fastq v2.17 for Novaseq output.
ATAC-seq reads were trimmed from 3' end until the final base had a quality score > 30, using Trimmomatic v0.33, discarding reads left with < 20 bp
ATAC-seq reads were aligned to the UCSC GRCm38 genome using Bowtie version 1.1.2. Only uniquely mapping reads with at most two mismatches were retained.
Peaks were called uing MACS v2.1.0 with the significance cut-off q-value <=0.05
bigWig files were generated using the bamCoverage. narrowPeak files were generated using MACS v2 with default settings.
Assembly: GRCm38
Supplementary files format and content: narrowPeak

source_name: primary murine osteosarcoma derived cell lines

growth_protocol: All cell lines were grown in DMEM with 10% FBS and 1% Penicillin/Streptomycin.

extract_protocol:
Bulk ATAC-Seq analysis was performed following the protocol of Buenrostro et al 2015. Tn5 transposase-tagmented genomic DNA was utilized to make libraries for next generation sequencing.
Bulk ATAC Sequencing Libraries were constructed following standard Illumina protocols for paired-end sample indexing with I7 and I5 sequencing indices.

tag: tissue

value: Murine osteosarcoma cell line

value: Osteosarcoma


title: Osteosarcoma from p53fl/fl group 471

description: Osteosarcoma from p53fl/fl group


title: Murine osteosarcoma cell line expressing p53R245W/- Gla_941_R2

value: primary murine osteosarcoma derived cell lines


title: Osteosarcoma from p53R245W/fl group 1818

description: Osteosarcoma from p53R245W/fl group


title: Osteosarcoma from p53fl/fl group 400

description: Osteosarcoma from p53fl/fl group


title: pbmc LRBA patient [P4_LRBA]

first: Mickael

middle: Mathieu

last: Menager

address: 24 Boulevard du Montparnasse

institute: Institut Imagine

data_processing:
Sequencing reads were demultiplexed and aligned to the human reference genome (GRCh38, release 98, built from ensembl sources), using the CellRanger Pipeline v3.1.0
Unfiltered RNA UMI counts were loaded into Seurat v4.0.1 (Stuart et al., 2019) for quality control, data integration and downstream analyses. Apoptotic cells and empty sequencing capsules were excluded by filtering out cells with fewer than 500 features or a mitochondrial content higher than 20%.
Data from each sample were SCT normalisedand scaled, before batch correction using Seurat’s FindIntegratedAnchors.
On this integrated dataset, we computed the principal component analysis on the 3000 most variable genes. UMAP was carried out using the 30 most significant PCs, and community detection was performed using the graph-based modularity-optimization Louvain algorithm from Seurat’s FindClusters function with a 1.6 resolution.
Cell types labels were assigned to resulting clusters based on a manually curated list of marker genes as well as previously defined signatures of the well-known PBMC subtypes (Monaco et al., 2019).  134776 cells were kept for further analysis.
Genome_build: GRCh38, release 98
Supplementary_files_format_and_content: h5 files contain the raw count matrix for each sample and a rds file containing the integrated dataset.

molecule: polyA RNA

source_name: pbmc LRBA patient

extract_protocol:
The scRNA-seq libraries were generated using 10X Chromium Single Cell 3′ Library & Gel Bead Kit (v.2 for batch 4, v.3 for batch 1 and 2 and v.3.1. for batch 3) according to the manufacturer’s protocol. Cells were counted, diluted at 1000 cells/µL in PBS+0,04% and 20 000 cells were loaded in the 10x Chromium Controller to generate single-cell 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 26 cycles of read 1, 8 cycles of i7 index and 98 cycles of read 2 (for kit v.2.) or with 28 cycles of read 1, 8 cycles of i7 index and 91 cycles of read 2 (for kit v.3 and v.3.1).

value: pbmc

tag: group

value: LRBA

tag: batch

value: Batch 3

value: no

tag: age

tag: gender

value: F

tag: mutated allele

value: homozygote


title: pbmc NBEAL2 patient [P5.II-1_NBEAL2]

source_name: pbmc NBEAL2 patient

tag: cell type


title: Murine osteosarcoma cell line expressing p53R245W/- Gla_941_R1

tag: cell line


title: Osteosarcoma from p53R245W/fl group 1813

description: Osteosarcoma from p53R245W/fl group

value: Primary osteosarcoma


title: pbmc NBEAL2 patient [P8_NBEAL2]

tag: treatment


title: Osteosarcoma from p53fl/fl group 459

description: Osteosarcoma from p53fl/fl group

tag: genotype


title: Murine osteosarcoma cell line expressing p53-/- Gla_561_R3


title: Osteosarcoma from p53fl/fl group 6019

description: Osteosarcoma from p53fl/fl group


title: pbmc LRBA patient [P2_LRBA]

value: Batch 2


title: Osteosarcoma from p53R245W/fl group 408

description: Osteosarcoma from p53R245W/fl group


title: Murine osteosarcoma cell line expressing p53-/- Gla_561_R2


title: Osteosarcoma from p53R245W/fl group 1575

description: Osteosarcoma from p53R245W/fl group


title: pbmc NBEAL2 patient [P7_NBEAL2]

value: M


title: Osteosarcoma from p53fl/fl group 486

description: Osteosarcoma from p53fl/fl group


title: pbmc LRBA patient [P3_LRBA]


title: Osteosarcoma from p53R245W/fl group 1827

description: Osteosarcoma from p53R245W/fl group


title: Osteosarcoma from p53R245W/fl group 555

description: Osteosarcoma from p53R245W/fl group


title: Osteosarcoma from p53R245W/fl group 494

description: Osteosarcoma from p53R245W/fl group


title: Osteosarcoma from p53R245W/fl group 1833

description: Osteosarcoma from p53R245W/fl group


title: Murine osteosarcoma cell line expressing p53R172H/- Gla_424_R2


title: Osteosarcoma from p53R245W/fl group 485

description: Osteosarcoma from p53R245W/fl group


title: pbmc NBEAL2 patient [P2_NBEAL2]

value: Batch 1


title: Osteosarcoma from p53R245W/fl group 433

description: Osteosarcoma from p53R245W/fl group


title: Osteosarcoma from p53R245W/fl group 1832

description: Osteosarcoma from p53R245W/fl group


title: Osteosarcoma from p53R245W/fl group 431

description: Osteosarcoma from p53R245W/fl group


title: pbmc LRBA patient [P1.II-1_LRBA]


title: pbmc LRBA patient [P1.II-2_LRBA]


title: EH.2

city: Philadelphia

first: Priyankara

middle: J

last: Wickramasinghe

state: PA

address: 3601 Spruce Street

department: Bioinformatics

institute: The Wistar Institute

data_processing:
RNA-seq data were aligned using STAR algorithm against mm10 mouse genome version and RSEM v1.2.12 software was used to estimate read counts and FPKM values using gene information from the Ensemble transcriptome version GRCm38.89
Assembly: mm10
Supplementary files format and content: RSEM raw counts for every gene

source_name: mammary gland

extract_protocol:
RNA from lysed mammary gland tissue was extracted using the Direct-zol RNA Miniprep kit (Zymo Research, Irvine, CA) according to the manufacturer’s instructions.
Libraries for whole transcriptome RNA-Seq were prepared using the Sense mRNA-Seq Library Prep Kit V2 (Lexogen, Vienna, Austria) according to the manufacturer’s instructions starting with an input of 300 ng of RNA and 13 cycles of final PCR amplification.

value: mammary gland

value: hypoxia exosomes


title: EH.1


title: Osteosarcoma from p53R245W/fl group 1831

description: Osteosarcoma from p53R245W/fl group


title: Osteosarcoma from p53fl/fl group 327

description: Osteosarcoma from p53fl/fl group


title: Osteosarcoma from p53fl/fl group 562

description: Osteosarcoma from p53fl/fl group


title: Murine osteosarcoma cell line expressing p53-/- Gla_561_R1


title: EN.3

value: normoxia exosomes


title: pbmc control [C6]

source_name: pbmc control

value: Control

value: wt


title: Osteosarcoma from p53R172H/fl group 424

description: Osteosarcoma from p53R172H/fl group


title: Osteosarcoma from p53fl/fl group 1979

description: Osteosarcoma from p53fl/fl group


title: Murine osteosarcoma cell line expressing p53R172H/- Gla_424_R3

value: Murine osteosarcoma cell line

value: Osteosarcoma


title: EN.2


title: pbmc control [C4]

value: pbmc


title: Osteosarcoma from p53R245W/fl group 941

description: Osteosarcoma from p53R245W/fl group


title: pbmc NBEAL2 patient [P4.II-1_NBEAL2]


title: pbmc control [C3]

value: Batch 4


title: Osteosarcoma from p53R172H/fl group 344

description: Osteosarcoma from p53R172H/fl group


title: Osteosarcoma from p53R172H/fl group 470

description: Osteosarcoma from p53R172H/fl group


title: Osteosarcoma from p53fl/fl group 1522

description: Osteosarcoma from p53fl/fl group


title: Osteosarcoma from p53R245W/fl group 1835

description: Osteosarcoma from p53R245W/fl group


title: Murine osteosarcoma cell line expressing p53R172H/- Gla_424_R1

value: primary murine osteosarcoma derived cell lines


title: Osteosarcoma from p53fl/fl group 1640

description: Osteosarcoma from p53fl/fl group


title: pbmc control [C27]

value: Batch 3

value: no


title: ctrl.3

value: control


title: pbmc control [C26]

value: F


title: Osteosarcoma from p53fl/fl group 1606

description: Osteosarcoma from p53fl/fl group

value: Primary osteosarcoma


title: ctrl.1


title: Osteosarcoma from p53fl/fl group 1512

description: Osteosarcoma from p53fl/fl group


title: ctrl.2


title: EN.1


title: tissue, epithelial cells, rep 2

city: Berlin

first: Michael

last: Sigal

address: Augustenburger Platz 1

department: Dept. Hepatology and Gastroenterology

country: Germany

institute: Charité Universitätsmedizin

description: Epithelial cells from colon tissue sample, replicate 2

library_source: transcriptomic single cell

data_processing:
The demultiplexing, barcoded processing, gene counting and aggregation were made using the Cell Ranger software v6  for the stromal cell samples and v7.1.0 for the epithelial samples (https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/what-is-cell-ranger)
Assembly: mm10
Supplementary files format and content: Filtered matrices from CellRanger

source_name: colon tissue

extract_protocol:
Libraries were constructed using Single Cell 3' Library & Gel Bead Kit v3 and the A Chip Kit (10x genomics) according to the manufacturer's instructions. Briefly, cells were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM).
Library was performed according to the manufacter’s instructions (Single Cell 3' v3 Gene Expression protocol, 10x Genomics)

value: colon tissue

value: epithelial


title: organoids, epithelial cells, rep 2

description: Epithelial cells from colon organoids, replicate 2

source_name: colon organoids

value: colon organoids


title: assembloids, epithelial cells, rep 2

description: Epithelial cells from colon assembloids, replicate 2

source_name: colon assembloids

value: colon assembloids


title: MZ_290_WT

city: Kansas City

first: Huzaifa

last: Hassan

state: MO

institute: Stowers Institute for Medical Research

description: Libraries were normalized, pooled, multiplexed, and sequenced on an Illumina NextSeq 500  with 75bp single read runs.

data_processing:
Raw reads were demultiplexed into Fastq format allowing up to one mismatch using Illumina bcl-convert 3.10.5 Reads were aligned to UCSC genome mm10 with STAR aligner (version 2.7.3a ), using Ensembl 102 gene models. TPM values were generated using RSEM (version v1.3.0 )
Assembly: mm10
Supplementary files format and content: TPM gene expression in text file

source_name: Placenta

extract_protocol:
Total RNA from TGCs was isolated using TRIzol (#15596026, Thermo Fisher Scientific).
rRNA was depleted using NEBNext rRNA Depletion Kit (Human/Mouse/Rat) (NEB # E6310L). cDNA synthesis was performed using NEBNext Ultra II Directional RNA Library Prep Kit (NEB #E7760S) and NEBNext Multiplex Oligos for Illumina (Index Primer Set 1) (NEB # E7335S). Sequencing was performed using an Illumina NextSeq 500  with 75bp single read runs.

value: Placenta

value: Trophoblast cells

value: Wild type


title: Liver_1_HTO

city: Madrid

first: Rui

last: Benedito

state: Madrid

address: C. de Melchor Fernández Almagro, 3

country: Spain

institute: CNIC

description: 10x Genomics

data_processing:
Liver1_GEX_Dll4iDEC (BC304), Notch1iDEC (BC302) and RbpjiDEC (BC305) groups data were shown in UMAPs of Figure 4, 6, 7 and corresponding Extended Data Figures. Also used for violin/dot plots or heatmaps shown in these figures. Data from control groups of Liver1_GEX (BC303) and Liver2_GEX (BC301) were merged and displayed in UMAPs of Figures 4, 5, 6, 7 and corresponding Extended Data Figures. Also used for related violin/dot plots or heatmaps shown in these figures.
The demultiplexing, barcoded processing, gene counting and aggregation were made using the Cell Ranger software v4.0 or v6.1 (https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/what-is-cell-ranger)
For alignment and quantification of gene expression the reference transcriptome was built using mouse genome GRCm38 and ensembl gene build version 98 (sep2019.archive.ensembl.org). The phiYFP-sv40pA, MbTomato-2A-Cre-WPRE-sv40pa or CreERT2 transgenes sequences expressed in the samples were added to the reference
Liver ECs: The following quality control steps were performed to minimize low-quality cells and improve posterior normalization and analysis: i) A minimum of normalized counts per cell of 1500 and a maximum of 40000 was applied. ii) A minimum gene detection filter of 600 genes. iii) A maximum 25% of MT content. iv) A minimum cell fraction in the sample of 0.1%. The cell fraction refers to the proportion of UMIs the cell contributes with to the total UMIs in the sample. v) Cells with high numbers of reads in just a few genes were filtered out. vi) Cells with more than 0.1% reads in HBB genes were removed. vii) Cells with < 100 UMIs in the hashtags were removed to improve sample calling. Viii) Only single cells were selected; doublets were filtered out.
Cells were de-multiplexed using the pipeline provided with the Seurat package. Counts were normalized and scaled for posterior analysis. Clusters were identified using the nearest neighbors algorithm at different resolutions. Clusters and cells were classified based on the SingleR method using Blueprint Encode and the Human Primary Cell Atlas cell type profiles collection
Following cluster identification with the starting dataset, the remaining Liver EC datasets were mapped using the FindTransferAnchors() function from the Seurat package using 30 PCA dimensions with the default settings.
Liver non ECs: Cells were de-multiplexed by aplying the cellranger multi pipeline. The following quality control steps were performed to minimize low-quality cells and improve posterior normalization and analysis: i) A minimum of normalized counts per cell of 2000 and a maximum of 30000 was applied. ii) A minimum gene detection filter of 500 genes and a maximum of 6000. iii) A maximum 5% of MT content. iv) A maximum 35% ribosomal content. v) a maximum 1% of Hbb content. Viii) Only single cells were selected; doublets were filtered out in the cellranger multi demultiplexing step.
Counts were log-normalized and scaled followed by PCA and clustering using the shared nearest neighbours algorithm and Louvain clustering (settings as defaults except for the 2000 most variable genes, 30 principal components and a resolution of 0.8). Clusters and cells were classified based on the Seurat method using Blueprint Encode, Human Primary Cell Atlas and mouseRNAseq datasets available in the celldex package, as well as a recent dataset from Ben-Moshe et al 2019 in order to classify each cluster to a different cell type. Final clustering was later manually refined based on markers expression.
Assembly: mm10
Supplementary files format and content: fasta file
Supplementary files format and content: gtf file
Supplementary files format and content: csv expression matrix
Supplementary files format and content: csv metadata

source_name: Liver

extract_protocol:
To isolate ECs for scRNAseq experiments, livers were dissected, minced, and digested for 30 min with pre-warmed (37C) dissociation buffer (2.5mg/ml collagenase I (Thermo Fisher 17100017 ), 2.5mg/ml dispase II (Thermo Fisher 17105041), 1 μl /ml DNAse in PBS containing Ca2+ and Mg2+ (Gibco)). The digestion tube was agitated every 3-5 minutes in a water bath. At the end of the 30 minutes incubation, sample tubes were filled up to 15 ml with sorting buffer (PBS containing no Ca2+ or Mg2+ and supplemented with 10% FBS (Sigma, F7524)) and centrifuged (450g, 5 min, 4°C). The supernatant was aspirated, and cell pellets were resuspended in 2ml 1x RBC lysis buffer (BioLegend, 420301) and incubated for 5 min on ice. To each sample were added 6 ml of sorting buffer, and samples were then passed through a 70um filter. Live cells were counted in a Neubauer Chamber using trypan blue exclusion. Cells from each condition (4x10^6/condition) were collected in separate tubes, and cells were incubated for 30 min with horizontal rotation in 300 μl antibody incubation buffer (PBS + 1% BSA) containing 1 μl CD31-APC, 1 μl CD45-APC-Cy7, and 1μl of hash tag oligo (HTO) conjugated antibodies (Biolegend). HTOs were used to label and distinguish the different samples when loaded on the same 10x genomics port, and in this way also guarantee the absence of batch effects. After the antibodies incubation, samples were transferred to 15 ml Falcon tubes, 10 ml sorting buffer was added, and samples were centrifuged (450g, 5min, 4°C). The supernatant was aspirated, pellets were resuspended in 1.5 ml sorting buffer and transferred to Eppendorf tubes, and the resulting suspensions were centrifuged (450g, 5min, 4°C). The resulting pellets were resuspended in 300 μl sorting buffer containing DAPI. Cells were sorted by FACS with an Aria Cell Sorter (BD Biosciences); ECs were identified as CD31+, CD45- MbTomato+ cells. After sorting, cells were centrifuged at 500g, 5min, 4ºC and the cell pellet was resuspended in cell capture buffer (PBS + 0.04% ultra-pure BSA (Thermo Fisher, AM2616) in order to get a concentration of 700-1200 cells/μl.
Single cell suspensions of freshly isolated ECs were resuspended in Cell Capture buffer to a final concentration of 800 to 1600 cells/μl. scRNA-seq libraries were prepared using the Chromium Single Cell 3´ Reagent Kits v3.1 (10x Genomics; Pleasanton, CA, USA) according to the manufacturer’s instructions.

treatment_protocol:
1.5 mg tamoxifen was injected for 4 consecutive days in each mouse. Livers of mice were processed on day 14 after the first tamoxifen injection.
Genotypes of experimental groups were: Control (Tg(Cdh5-CreERT2), Tg(iSuRe-Cre)); Dll4iDEC (Dll4flox/flox, Tg(Cdh5-CreERT2), Tg(iSuRe-Cre); Notch1iDEC (Notch1flox/flox, Tg(Cdh5-CreERT2, Tg(iSuRe-Cre); RbpjiDEC (Rbpjflox/flox, Tg(Cdh5-CreERT2, Tg(iSuRe-Cre);

value: Endothelial cells

value: Liver

tag: strain

tag: library type

value: HTO

value: Cdh5-CreERT2 + iSuRe-Cre

value: 10-20 weeks old

tag: hto demultiplexing_conditions


title: MZ_294_WT


title: organoids, epithelial cells, rep 1

description: Epithelial cells from colon organoids, replicate 1


title: assembloids, epithelial cells, rep 1

description: Epithelial cells from colon assembloids, replicate 1


title: pbmc control [C1]

value: pbmc

value: Control

value: Batch 1

value: wt


title: Liver_1_GEX_CD31+_ECs_scRNASeq_Control_Dll4iDEC_Notch1iDEC_RbpjiDEC

description: 10x Genomics

value: mRNA


title: MZ_283_WT


title: assembloid culture, stromal cells, rep 2

description: Stromal cells from colon assembloid cultures, replicate 2

value: stromal


title: Osteosarcoma from p53fl/fl group 1427

description: Osteosarcoma from p53fl/fl group

source_name: Primary osteosarcoma

value: Primary osteosarcoma


title: MZ_282_WT


title: s20

city: Jacksonville

first: Yingxue

last: Ren

state: FL

address: 4500 San Pablo Rd S

institute: Mayo Clinic

data_processing:
RNA samples were sequenced with Illumina HiSeq 4000 at the Mayo Clinic Genome Facility. Reads were mapped to the mouse genome mm10.
Raw gene read counts and sequencing quality control were generated using the Mayo Clinic RNA-sequencing analytic pipeline, MAP-RSeq Version 3.0
We performed conditional quantile normalization (CQN) on raw gene counts to correct for GC bias and gene length differences, and to also obtain similar quantile-by-quantile distributions of gene expression levels across samples.
Assembly: mm10
Supplementary files format and content: tab-delimited text file of combined gene counts of all samples

source_name: cortex

extract_protocol:
Total RNAs were extracted from cortical  samples using TRIzol RNA Isolation Reagents (Thermo Fisher Scientific) and a RNeasy mini kit with DNase (QIAGEN).
RNA libraries were prepared using the TruSeq RNA Sample Prep Kit v2 (Illumina, San Diego, CA) according to the manufacturer’s instructions.

value: cortex

value: APOE4; smLrp1-/-


title: MZ_280_WT

value: Placenta


title: s19


title: MZ_296_KO

value: Trophoblast cells

value: c-Myc null


title: s18


title: pbmc control [C2]

value: no

value: M


title: assembloid culture, stromal cells, rep 1

description: Stromal cells from colon assembloid cultures, replicate 1

value: colon assembloids


title: Osteosarcoma from p53fl/fl group 1431

description: Osteosarcoma from p53fl/fl group


title: MZ_289_KO


title: s17


title: tissue, epithelial cells, rep 1

description: Epithelial cells from colon tissue sample, replicate 1

value: colon tissue

value: epithelial


title: MZ_281_KO


title: Amp_Cas9_HBG_vivo_rep2

description: amplicon

source_name: HEK293T cells

value: embryonic kidney cells

tag: tissue source

value: kidney

tag: sample type

value: deep sequencing

tag: guide rna

value: treat(HBG-sgRNA); control (no gRNA)

tag: vivo or vitro

value: in vivo


title: Amp_Cas9_HBG_vivo_rep1

description: amplicon

tag: editor


title: MZ_277_KO

tag: tissue


title: s15

value: APOE4; control


title: Tumor bearing_TCR

city: Bethesda

first: Tim

middle: F.

last: Greten

state: MD

address: 10 Center Drive, Building 10

department: National Cancer Institute (NCI)

institute: National Institutes of Health

description: 10x Genomics TCR

data_processing:
Base calling was performed using RTA 3.4.4, demultiplexing was performed using cellranger v6.0.0 (Bcl2fastq 2.20.0), and alignment was performed using cellranger v6.0.0 (STAR 2.7.2a). Sequenced reads were aligned to the 10x Genomics provided mouse reference sequences (refdata-gex-mm10-2020-A for GEX and refdata-cellranger-vdj-GRCm38-alts-ensembl-5.0.0 for TCR).
Assembly: refdata-gex-mm10-2020-A for GEX and refdata-cellranger-vdj-GRCm38-alts-ensembl-5.0.0 for TCR
Supplementary files format and content: .h5ad: count matrix files in the Hierarchical Data Format (HDF5 or H5). Contains the combined processed data for both GEX and TCR sequencing.

source_name: Liver + tumor

growth_protocol: RIL-175 cells were maintained in RPMI supplemented with 10% fetal bovine serum (FBS) and antibiotics in humidified atmosphere with 5% CO2 at 37C prior to intrahepatic injection of 200000 cells/mouse

extract_protocol:
Hepatic MNCs were isolated and subjected to FACS based on CD45 expresion
Library was performed according to the manufacter’s instructions (single cell 5' v2 and VDJprotocol, 10x Genomics). Briefly, GCs were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM). The poly-A RNA from the cell lysate contained in every single GEM was retrotranscripted to cDNA, which contains an Ilumina R1 primer sequence, Unique Molecular Identifier (UMI) and the 10x Barcode. The pooled barcoded cDNA was then cleaned up with Silane DynaBeads, amplified by PCR and the apropiated sized fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction Ilumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added.
RNA libraries for RNA-seq were prepared using SMARTER mRNA-Seq Library Prep Kit following manufacturer's protocols.

value: Liver + tumor

value: Hepatic CD45+ leukocytes

value: 7 weeks old


title: inactive ASO, SB28 glioblastoma, rep2

city: St. Louis

first: Albert

last: Kim

address: 660 S. Euclid Avenue

department: Department of Neurological Surgery

institute: Washington University School of Medicine

data_processing:
The demultiplexing, barcoded processing, gene counting and aggregation were made using the Cell Ranger software v7.0.1
Assembly: mm10
Supplementary files format and content: Tab-separated values files and matrix files

source_name: glioblastoma

growth_protocol: Mice were aged to 3-5 months; Mouse pups were postnatal day 2.

extract_protocol:
Total RNA was extracted from BMDMs and microglia using QIA shredder (Qiagen, 79656) and RNeasy Plus Mini Kit (Qiagen, 74134), following the manufacture's instructions. Single-cell suspensions were made using Brain Tumor Dissociation Kit (Miltenyi Biotec, 130-095-942), CD45+ cell populations were enriched using CD45 MicroBeads (Miltenyi Biotec, 130-052-301).
Library construction was done following manufacturer protocol (10X Genomics Single Cell 3' v3)

treatment_protocol: One single dose of ASO intracerebroventricular injection; SB28 tumor cell intracerebral injection (8,000 cells/mouse)

value: glioblastoma

value: brain tumor

value: CD45+/CD45- cells

value: 400 μg inactive ASO


title: s14

value: cortex


title: Tumor bearing_GEX

description: 10x Genomics 5' v2


title: Pooled Aroclor1260 + PCB126, negative IgG IP

city: Louisville

first: Eric

middle: Christian

last: Rouchka

state: Kentucky

address: 522 East Gray Street

department: Biochemistry and Molecular Genetics

institute: University of Louisville

data_processing:
Base calling was performed with bcl2fastq v2.20.0.422.
Sequences were assessed for quality control using FastQC v0.10.1.
Low quality and sequence adapters were trimmed using Trimmomatic.
Sequences were aligned to the mm10 genome using bwa mem v0.7.17 with additional parameters -k 18 -T 18.
Peak calling was performed using MeTPeak v1.1.
Assembly: mm10
Supplementary files format and content: Tab-delimited MetPeak detected peaks
Library strategy: MeRIP-seq

source_name: liver

extract_protocol:
Trizol, Universal Plus mRNA-Seq kit standard protocol (Tecan Genomics), Magna-Me-RIP m6A kit (Millipore Sigma)
Libraries were prepared using the Ovation SoLo RNA-Seq Systems (NuGEN Cat# 0500-32).

treatment_protocol: Male C57Bl/6J mice were fed a HFD (42% kCal from fat) and treated with a single exposure of oral Aroclor 1260, PCB 126, and the combination of Aroclor 1260 + PCB 126 at 2 wks and the study contintued for a total of 12 weeks.

value: liver

tag: Sex

value: male

tag: group

value: IP


title: Amp_Cas9_HBG_vitro_rep3

description: amplicon

value: embryonic kidney cells


title: inactive ASO, SB28 glioblastoma, rep1


title: Pooled PCB126, negative IgG IP


title: s13


title: Amp_Cas9_HBG_vitro_rep2

description: amplicon

value: kidney


title: s12


title: Trem2 ASO, SB28 glioblastoma, rep1

value: 400 μg Trem2 ASO


title: Tumor free_TCR

description: 10x Genomics TCR

value: Liver


title: Cas9_nogRNA_vitro_IP

value: no

value: in vitro


title: Pooled  control, negative IgG IP

value: Control


title: Amp_Cas9_HBG_vivo_rep3

description: amplicon

value: deep sequencing

value: treat(HBG-sgRNA); control (no gRNA)

value: in vivo


title: s16

value: APOE4; smLrp1-/-


title: Tumor free_GEX

description: 10x Genomics 5' v2

value: Hepatic CD45+ leukocytes

value: 7 weeks old


title: s11

value: cortex

value: APOE4; control


title: Cas9_nogRNA_vitro_In

value: input


title: s10

value: APOE3; smLrp1-/-


title: cortex, Trem2, tumor antigen, rep2

data_processing:
Basecalls and demultiplexing were performed with Illumina’s bcl2fastq software with a maximum of one mismatch in the indexing read.
RNA-seq reads were then aligned to the Ensembl release 101 primary assembly with STAR version 2.7.9a1.
Gene counts were derived from the number of uniquely aligned unambiguous reads by Subread:featureCount version 2.0.32.
Isoform expression of known Ensembl transcripts were quantified with Salmon version 1.5.23.
Sequencing performance was assessed for the total number of aligned reads, total number of uniquely aligned reads, and features detected.  The ribosomal fraction, known junction saturation, and read distribution over known gene models were quantified with RSeQC version 4.04.
Assembly: mm10
Supplementary files format and content: Tab-delimited text files with Count, CPM, and FPKM
Supplementary files format and content: Tab-delimited text file with isoform TPM values

source_name: Brain cortex

extract_protocol:
Total RNA was extracted from BMDMs and microglia using QIA shredder (Qiagen, 79656) and RNeasy Plus Mini Kit (Qiagen, 74134), following the manufacture's instructions. Single-cell suspensions were made using Brain Tumor Dissociation Kit (Miltenyi Biotec, 130-095-942), CD45+ cell populations were enriched using CD45 MicroBeads (Miltenyi Biotec, 130-052-301).
After determining the integrity on an Agilent Bioanalyzer, extracted total RNA was prepared through Ribo-depletion for mRNA-seq

value: Brain cortex

value: primary cell

value: Microglia

value: Trem2 ASO; dead SB28 cells


title: MZ_279_KO

value: Placenta

value: Trophoblast cells

value: c-Myc null


title: cortex, Trem2, tumor antigen, rep1


title: High fat diet, Aroclor1260 + PCB126 m6A, replicate 4

value: liver

value: male


title: s9


title: BE3_nogRNA_vitro_IP

data_processing:
Quality of all reads was inspected using FastQC. Low-quality and adapter-containing reads were trimmed from raw data using Cutadapt. Then, the filtered reads were mapped to the reference human genome (hg19) using the bowtie2 aligner with default settings. The SAM files were subsequently converted to the BAM file format, sorted and indexed using SAMtools. Duplicate reads were removed using picard-tools.
Peaks were called using MACS (version 2.1.2) with default settings (-p 0.01). No-sgRNA (CRISPR alone) and GFP samples were used as negative control. Common peaks between the edited and control sample were removed by BEDtools intersect command. We then compared the results with ENCODE DAC Exclusion List Regions (ENCODE #: ENCSR636HFF) to filter out artifacts using BEDtools intersect command. The rest of peaks in edited sample were candidates of off-target sites.
CROss-seq sequencing data was analyzed using custom computational pipeline. The scoring program used for capturing off-target cleavage sites or editing sites have illustrated in paper.
Genome_build: hg19
Supplementary_files_format_and_content: XLS files include peaks for each sample, TDF files for loaded in IGV for visualization

value: embryonic kidney cells

value: kidney

value: IP

value: no

value: in vitro


title: Cas9_HEK2_vitro_IP


title: s7

value: cortex


title: High fat diet, Aroclor1260 + PCB126 m6A, replicate 3


title: Amp_Cas9_HBG_vitro_rep1

description: amplicon

value: deep sequencing

value: treat(HBG-sgRNA); control (no gRNA)

value: in vivo


title: cortex, Trem2, IFNγ, rep1

value: Brain cortex

value: primary cell

value: Trem2 ASO; IFNgamma


title: s8

value: APOE3; smLrp1-/-


title: BE3_HEK2_vitro_IP


title: s6


title: Pooled Aroclor1260, negative IgG IP


title: High fat diet, Aroclor1260 + PCB126 m6A, replicate 2


title: High fat diet, Aroclor1260 + PCB126 m6A, replicate 1


title: ABE7.10_nogRNA_vitro_IP


title: High fat diet, PCB126 m6A, replicate 5


title: cortex, Trem2, IFNγ, rep2

value: Microglia


title: Trem2 ASO, SB28 glioblastoma, rep2

value: glioblastoma

value: brain tumor

value: CD45+/CD45- cells

value: 401 μg Trem2 ASO


title: cortex, Trem2, PBS, rep1

value: Trem2 ASO; PBS


title: s4

value: APOE3; control


title: High fat diet, Aroclor1260 + PCB126 m6A, replicate 5

value: liver

value: male


title: cortex, inactive, tumor antigen, rep1

value: inactive ASO; dead SB28 cells


title: High fat diet, PCB126 m6A, replicate 4


title: cortex, inactive, tumor antigen, rep2


title: s3

value: cortex


title: 36_h_alsS_UB_2

city: Chicago

first: Peter

last: Tran

state: Illinois

address: impson Querrey Research Bldg 302 E Huron St, Room 11-500

institute: Prindle Lab, Northwestern University

description:
PT-23
Replicate 2

data_processing:
FastQC
Reads were trimmed to remove Illumina adapters from the 3’ ends using cutadapt
Trimmed reads were aligned to the Bacillus subtilis genome strain 3610 NCBI CP020102.1 and plasmid NCBI CP020103.1 using STAR
Read counts for each gene were calculated using htseq-count (Anders et al, 2015) in conjunction with a gene annotation file for the reference genomes obtained from NCBI
Normalization and differential expression were calculated using DESeq2 that employs the Wald test (Love et al, 2014)
Assembly: Bacillus subtilis genome strain 3610 NCBI CP020102.1 and plasmid NCBI CP020103.1 using STAR
Supplementary files format and content: tab-delimited text files include RPKM values for each Sample

organism: Bacillus subtilis

source_name: Biofilm grown for 36 h

growth_protocol: Biofilms were grown on MSgg agar for 24 to 36 h.

extract_protocol:
Biofilms were harvested into 80% methanol and sonciated, followed by clean-up and DNase I treatment with QIAGEN RNeasy mini kit in accordance with the prescribed protocol provided with the kit. Quality control was performed with Agilent Bioanalyser.
RNA libraries for RNA-seq were prepared using Illumina® Stranded TotalRNAPrep, Ligation with Ribo-Zero Plus Kit following manufacturer's protocols.

treatment_protocol: Biofilms were grown on either MSgg with 100 mM or 1 mM MOPS buffer.

value: NCIB 3610

value: Gram-positive bacteria

value: {delta}alsS

value: Minimally buffered


title: 36_h_alsS_UB_3

description:
PT-24
Replicate 3


title: ABE7.10_HEK2_vitro_IP

extract_protocol:
Genomic DNA was freshly extracted using FastPure Cell/Tissue DNA Isolation MiniKit and stored in 25 mM K3BO3 buffer (pH 7.0) at -20 °C. Treated genomic DNA was  purified by DNeasy Blood & Tissue Kit and eluted by  25 mM K3BO3 buffer (pH 7.0).
For library preparation, fragmented DNA first be repaired and ligated to adapters, then, the IP sample was subjected to biotin enrichment using Dynabeads MyOne Streptavidin C1 (Thermo, 65001). All reactions were followed the protocol of VAHTS Universal DNA Library Prep Kit for Illumina V3 (Vazyme, ND607) with some modifications. The biotin pull-down experiments were performed following the recommendations from the Dynabeads MyOne Streptavidin C1 (Thermo, 65001) with some modifications.
CROss-seq (biotin enriched ChIP-seq), Amplicon

value: embryonic kidney cells

value: kidney

value: IP

value: in vitro


title: cortex, Trem2, PBS, rep2

value: Brain cortex

value: primary cell

value: Microglia


title: High fat diet, PCB126 m6A, replicate 3


title: 36_h_alsS_UB_1

description:
PT-22
Replicate 1


title: High fat diet, PCB126 m6A, replicate 2


title: s1


title: cortex, inactive, IFNγ, rep2

value: inactive ASO; IFNgamma


title: High fat diet, PCB126 m6A, replicate 1


title: s2

value: APOE3; control


title: cortex, inactive, PBS, rep2

value: inactive ASO; PBS


title: cortex, inactive, IFNγ, rep1


title: 36h_alsS_control_3

description:
PT-21
Replicate 3

value: Buffered


title: High fat diet, Aroclor1260 m6A, replicate 5

value: liver

value: male


title: 36h_alsS_control_2

description:
PT-20
Replicate 2

value: NCIB 3610


title: Embryonic endothelial cells - KO rep2

city: Charlottesville

first: Uma Devi

last: Paila

address: 1335 Lee St

institute: University of Virginia

data_processing:
Cell Ranger software pipeline (10X genomics) was used for processing the raw files
Seurat R package version 4 was used for downstream analysis
Phate software (Moon et.al., Nat Biotechnol., 2019) was used for dimensionality reduction and trajectory analysis
Genome_build: NOT PROVIDED
Supplementary_files_format_and_content: filtered feature-barcode matrices from the Cell Ranger output for each replicate (tsv and matrix files are contained in a folder for each replicate)

source_name: Endothelial cells

extract_protocol:
CD31+/C45- EC were FACS-sorted from E10.5 AGM of age-matched WT and miR-223-/- mutant into 0.04% BSA in PBS that had >85% viability
Sorted cells were processed for single cell mRNA profiling by barcoding with 10-X Genomics Chromium NextGEM Single Cell 3' library construction Kit v3 according to manufacturer instructions.

value: aorta-gonad-mesonephros region (AGM)

tag: age

value: embryonic day 10.5


title: s5

value: cortex


title: cortex, inactive, PBS, rep1


title: Embryonic endothelial cells - KO rep1


title: 36h_alsS_control_1

description:
PT-19
Replicate 1

value: Gram-positive bacteria

value: {delta}alsS


title: Trem2+/+, bone marrow, tumor antigen, rep2

source_name: Bone marrow

value: Bone marrow

value: macrophage

value: dead SB28 cells


title: High fat diet, Aroclor1260 m6A, replicate 4


title: High fat diet, Aroclor1260 m6A, replicate 3


title: Embryonic endothelial cells - WT rep2

value: wild type


title: 36h_WT_UB_3

description:
PT-18
Replicate 3

value: WT

value: Minimally buffered


title: Trem2+/+, bone marrow, tumor antigen, rep1

value: primary cell


title: 36h_WT_UB_2

description:
PT-17
Replicate 2


title: Trem2+/+, bone marrow, IFNγ, rep2

value: IFNgamma


title: Embryonic endothelial cells - WT rep1


title: High fat diet, Aroclor1260 m6A, replicate 2


title: 36h_WT_control_3

description:
PT-15
Replicate 3

value: Buffered


title: High fat diet, control m6A, replicate 5

value: liver

value: Control


title: 36h_WT_control_2

description:
PT-14
Replicate 2

value: NCIB 3610


title: Trem2+/+, bone marrow, IFNγ, rep1


title: 1 Day post-op nR181-L1-G7-P144

city: Irvine

first: Timothy

last: Downing

state: CA

address: 419 S. Circle View Drive

department: Department of Biomedical Engineering

institute: UCI

description:
1 Day post-op
RRBS

data_processing:
Quality and adapter trimming of raw fastq files was executed using Trim Galore!0.6.6 with the following parameters: “--paired --clip_r1 10 --clip_r2 15 --three_prime_clip_r1 10 --three_prime_clip_r2 10 --fastqc”.
Reads were aligned to the rat genome (mRatBN7.2, data: 2020/11/10) by using Bismark 0.23.0
On average, 22.8 million trimmed reads per sample were aligned to rat genome (mRatBN7.2, release 105, Ensembl).
CpG sites were called using the “bismark_methylation_extractor” function (-- comprehensive option) in Bismark
Differential methylation cytosines (DMCs) were called analysis using methylKit (1.14.2) for CpGs with a minimum of 5 counts
Assembly: mRatBN7.2

organism: Rattus norvegicus

source_name: esophageal tissue

extract_protocol:
DNA isolation was performed using Qiagen DNeasy Blood & Tissue kit (Qiagen) and genomic DNA was digested with MspI overnight. The digested products were size-selected using AMPure XP beads to remove anything above 300bp or small fragment retention.
DNA was bisulfite-converted using EZ DNA Methylation-Gold Kit (Zymo Research). The Accel-NGS Methyl-seq DNA library kit (Swift Biosciences) was used according to the manufacturer’s instructions.

value: esophageal tissue

value: 8-9 weeks

value: Male

tag: time

value: Day 1

value: biopsy punches were utilized to acquire host esophageal tissues surrounding 2 mm of the original defect circumference at 1 day post-op


title: Trem2+/+, bone marrow, LPS, rep2

value: Bone marrow

tag: cell type

value: LPS


title: 1 Day post-op nR181-L1-G7-P143

description:
1 Day post-op
RRBS


title: High fat diet, Aroclor1260 m6A, replicate 1

value: male


title: 1 Day post-op nR181-L1-G7-P141

description:
1 Day post-op
RRBS


title: 36h_WT_UB_1

description:
PT-16
Replicate 1

value: Gram-positive bacteria


title: 36h_WT_control_1

description:
PT-13
Replicate 1

value: WT


title: High fat diet, control m6A, replicate 4


title: 1 Day post-op nR181-L1-G7-P140

description:
1 Day post-op
RRBS


title: Trem2+/+, bone marrow, LPS, rep1

tag: cell line

value: macrophage


title: Trem2+/+, bone marrow, PBS, rep2

value: primary cell

tag: treatment

value: PBS


title: 24h_alsS_UB_3

description:
PT-12
Replicate 3

source_name: Biofilm grown for 24 h

tag: genotype

value: {delta}alsS

value: Minimally buffered


title: High fat diet, control m6A, replicate 2

value: liver

tag: strain

value: Control


title: 1 Day post-op nR181-L1-G7-P142

description:
1 Day post-op
RRBS

value: esophageal tissue


title: High fat diet, control m6A, replicate 3


title: Trem2+/+, bone marrow, PBS, rep1


title: Non-surgical control (NSC) nR181-L1-G7-P139

description:
Non-surgical control (NSC)
RRBS

value: 8-9 weeks

value: NSC

value: Full thickness esophageal specimens were harvested in parallel from NSC


title: High fat diet, control m6A, replicate 1


title: 24h_alsS_UB_2

description:
PT-11
Replicate 2

value: NCIB 3610


title: Trem2-/-, bone marrow, tumor antigen, rep2

value: Bone marrow

value: dead SB28 cells


title: Non-surgical control (NSC) nR181-L1-G7-P138

description:
Non-surgical control (NSC)
RRBS

value: Male


title: High fat diet, Aroclor1260 + PCB126 input, replicate 5

value: male

value: Input


title: Non-surgical control (NSC) nR181-L1-G7-P137

description:
Non-surgical control (NSC)
RRBS


title: Trem2-/-, bone marrow, tumor antigen, rep1

value: macrophage


title: 24h_alsS_control_3

description:
PT-09
Replicate 3

value: Gram-positive bacteria

value: Buffered


title: 24h_alsS_control_2

description:
PT-08
Replicate 2

value: {delta}alsS


title: 1 Wk post-op nR181-L1-G7-P135

description:
1 Wk post-op
RRBS

value: Day 7

value: neotissues at 1 week post-op were isolated from the initial graft area


title: Non-surgical control (NSC) nR181-L1-G7-P136

description:
Non-surgical control (NSC)
RRBS


title: Trem2-/-, bone marrow, IFNγ, rep1

value: primary cell

value: IFNgamma


title: 24h_alsS_control_1

description:
PT-07
Replicate 1


title: High fat diet, Aroclor1260 + PCB126 input, replicate 2

value: liver


title: Trem2-/-, bone marrow, IFNγ, rep2


title: High fat diet, Aroclor1260 + PCB126 input, replicate 1


title: 1 Wk post-op nR181-L1-G7-P133

description:
1 Wk post-op
RRBS

value: esophageal tissue

value: 8-9 weeks


title: 24h_WT_UB_3

description:
PT-06
Replicate 3

value: NCIB 3610

value: WT

value: Minimally buffered


title: Trem2-/-, bone marrow, LPS, rep1

value: Bone marrow

value: LPS


title: High fat diet, PCB126 input, replicate 5

value: male

value: Input


title: 1 Wk post-op nR181-L1-G7-P132

description:
1 Wk post-op
RRBS

value: Male


title: Trem2-/-, bone marrow, LPS, rep2

value: macrophage


title: Trem2-/-, bone marrow, PBS, rep2

value: PBS


title: 24h_WT_UB_1

description:
PT-04
Replicate 1

value: Gram-positive bacteria


title: 1 Wk post-op nR181-L1-G7-P134

description:
1 Wk post-op
RRBS

value: Day 7

value: neotissues at 1 week post-op were isolated from the initial graft area


title: High fat diet, PCB126 input, replicate 4


title: Trem2-/-, bone marrow, PBS, rep1

value: primary cell


title: 1 Wk post-op nR181-L1-G7-P131

description:
1 Wk post-op
RRBS


title: 24h_WT_UB_2

description:
PT-05
Replicate 2


title: 24h_alsS_UB_1

description:
PT-10
Replicate 1

value: {delta}alsS


title: 24h_WT_control_3

description:
PT-03
Replicate 3

value: Buffered


title: High fat diet, PCB126 input, replicate 3

value: liver


title: High fat diet, Aroclor1260 + PCB126 input, replicate 4


title: Wild type  replicate 2

description: 10x Genomics

data_processing:
Raw reads were demultiplexed into Fastq format using cellranger (v 3.0.0)  mkfastq. Alignment, filtering, barcode counting, and UMI counting was done using cellranger’s count pipeline
Assembly: mm10
Supplementary files format and content: Tab-separated values files and matrix files

extract_protocol: Chromium Next GEM Single Cell 3' Reagent Kits v3.1 was used for making cDNA library using 10X Genomic reagents.

value: Placenta

value: Trophoblast cells

value: Wild type


title: 24h_WT_control_2

description:
PT-02
Replicate 2

value: NCIB 3610

value: WT


title: High fat diet, PCB126 input, replicate 2

value: male


title: HAEC 2Gy rep2

city: Cheonan

first: Keunsoo

last: Kang

state: Chungnam

address: Dandae-ro 119

department: Microbiology

country: South Korea

institute: Dankook University

data_processing:
The raw RNA-seq reads acquired from the sequencing platform were processed using Trim Galore (https://www.bioinformatics.babraham.ac.uk/projects/trim_galore/) to eliminate adapter and low-quality sequences. The trimmed reads underwent quality assessment using FastQC (version 0.11.9) (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/).
The trimmed reads were aligned to the reference human genome (hg38 assembly; GRCh38.p13) using STAR (version 2.7.9a) (Dobin et al. Curr Protoc Bioinforma 2015) and GENCODE gene annotation (https://www.gencodegenes.org/). The RNA-seq data quality was further evaluated by calculating the transcript integrity number (TIN) score using RSeQC (tin.py; version 4.0.0) (Wang et al. Bioinforma Oxf Engl 2012). With a median TIN value exceeding 0.7 for all RNA-seq data, high data quality was confirmed.
The expression levels of known genes were estimated using the transcript per million (TPM) method with StringTie (version 2.1.7b) (Pertea et al. Nat. Biotechnol 2015). Differential expression analysis was performed using DESeq2 (version 1.36.0) (Love et al. Genome Biol 2014) with an adjusted p-value cutoff of 0.05. All software and algorithms were used with default parameters.
Assembly: hg38
Supplementary files format and content: Tab-delimited text files include TPM values for each Sample

source_name: HAEC

growth_protocol: Human aortic endothelial cells (HAECs, #CC-2535) were obtained from Lonza Group Ltd. (Walkersville, MD, USA). The cells were cultured in endothelial growth medium-2 microvascular medium (EGM-2MV, Lonza) and maintained at 37°C in a humidified incubator with 5% CO2.

extract_protocol:
Total RNA was isolated using the TRIsure solution (Bioline, London, UK) according to the manufacturer's instructions. RNA quality was assessed with an Agilent 2100 bioanalyzer and an RNA 6000 Nano Chip (Agilent Technologies, Amstelveen, Netherlands), obtaining the RNA integrity number. Total RNA quantification was performed using a NanoDrop 2000 spectrophotometer (ND-2000; Thermo Fisher Scientific, Inc.).
RNA-seq was conducted on high-quality RNA samples (RNA integrity number > 7) from HAEC cells exposed to 0Gy (no radiation), 0.1Gy, 0.5Gy, or 2.0Gy. Samples were multiplexed into lanes and sequenced using a HiSeq 4000 system (Illumina, San Diego, CA, USA).

treatment_protocol: Cells were exposed to γ-rays using a 137Cs laboratory γ-irradiator (LDI-KCCH 137, Seoul, Korea) at a dose of 0.1 Gy (4.8 mGy/min) or a 137Cs γ-ray source (Atomic Energy of Canada, Mississauga, Ontario, Canada) at doses of 0.5 Gy or 2 Gy (2.26 Gy/min), following a procedure described previously (Kaushik et al. Sci Rep 2017).

value: HAEC

value: human aortic endothelial cells


title: High fat diet, Aroclor1260 + PCB126 input, replicate 3

value: Input


title: HAEC 2Gy rep1


title: Wild type  replicate 1

description: 10x Genomics


title: High fat diet, PCB126 input, replicate 1


title: 24h_WT_control_1

description:
PT-01
Replicate 1


title: HAEC 0.5Gy rep2


title: High fat diet, Aroclor1260 input, replicate 5


title: ho-middle-rep1

city: Shanghai

first: Qing

address: 320 Yueyang Road

department: institute of biochemistry and cell biology

institute: Chinese Academy of Science

data_processing:
Raw RNA-seq reads were cleaned using fastp (v0.20.0) program, aligned to mm10 using STAR (v2.7.3a)
Only unique mapped reads were kept and counted by featureCounts (v2.0.0)
FPKM and TPM were calculated by stringtie (v2.0)
Genome coverage was counted by bamCoverage (v3.3.1)
Genome coverage was counted by bamCoverage (v3.3.1). Differential gene expression was carried out using DESeq2 (v1.30.0), and clusterProfiler (v3.18.1) was used for GO and KEGG analysis in R
Assembly: mm10
Supplementary files format and content: tab-delimited text files include RPKM values for each Sample

source_name: Cochlea

extract_protocol:
Total RNA of these sections was collected by miRNeasy Micro Kit (QIAGEN, Cat# 217084) following the manufacturer’s instructions.
All collected RNA was used for library construction through VAHTS Universal V8 RNA-seq Library Prep Kit for Illumina (Vazyme, Cat# NR605).
RNA-seq libraries subjected to deep sequencing on Illumina NovaSeq6000 platform following the manufacturer’s instructions (Illumina) to produce 150bp paired-end reads by Genergy Biotechnology Co. Ltd. (Shanghai, China).

value: Cochlea

tag: section

value: middle

value: KO


title: ho-middle-rep2


title: Primary lung E12.5

city: Boston

first: Pushpinder

last: Bawa

state: Massachusetts

address: 670 Albany St

department: Boston University Medical Campus

institute: Center for Regenerative Medicine

data_processing:
The sample was processed using the standard Cell ranger pipeline
Reads were mapped to GRCm38 with GFP and TdTomato
Downstream analysis and QC done on Seurat
Differential expression using MAST models
Assembly: GRCm38
Supplementary files format and content: h5 is the standard output from the cell ranger count pipeline

organism: Mus

source_name: C57/BL6J mouse embryonic lung mesenchyme and epithelium from day E12.5

extract_protocol:
Cells were resuspended in buffer at a concentration of 1000 cells/ul and captured using a 10X chromium instrument (10X Chromium, Pleasanton, CA), following standard Chromium 10x guidelines.
Libraries were prepared using a Chromium Single-Cell 30 Library Kit (10X Genomics) and loaded on a NextSeq500 (Illumina) with a custom sequencing setting to obtain a sequencing depth of ~40k reads/cell

treatment_protocol: All samples were sorted for live cells and single cells prior to extract preparation

tag: developmental stage

value: Embryonic day 12.5

tag: isolation method

value: FACS sorting for Epcam+ and Epcam- cells


title: cLM

source_name: "co-developed" day 13 Tbx4-LERGFP+ cells and Epcam+/Tbx4-LERGFP- generated by directed lung epithelial differentiation to simultaneously produce endoderm/epithelium and mesenchyme

growth_protocol: Cells were differentiated towards the lung epithelial progenitor state using our previously pulished protocol (Serra et al, Development 2015). Doxycyline was added to induce GFP+ lineage laeling of lung mesenchyme from day 6-13 of differentiation.

value: Day 13 of directed lung epithelial differentiation

value: FACS sorting for single, live, GFP+ and Epcam+/GFP- cells


title: High fat diet, Aroclor1260 input, replicate 4

value: liver


title: ho-base-rep2

last: Li

value: base


title: HAEC 0.1Gy rep2


title: High fat diet, Aroclor1260 input, replicate 3


title: ho-apex rep2

value: apex


title: iLMneg

source_name: Day 13 Tbx4-LER GFP- cells generated by directed lung mesenchymal differentiation

growth_protocol: iPSCs were differentiated towards the lateral plate mesoderm by 2 days of LIF withdrawal and subsequent treatment with recombinant Activin A, Bmp4 and Wnt3a for 3 days. Cells were then further differentiated towards the lung mesenchyme by adding retinoic acid and the Hedgehog agonist purmorphamine (PMA) for 8 days. Doxycycline was added to induce GFP+ lineage labeling of lung mesenchymal cells from day 5 - 13 of differentiation. Cells were differentiated in serum-free, feeder-free conditions.

value: Day 13 of directed lung mesenchymal differentiation

value: FACS sorting for single, live, GFP- cells


title: iLM

source_name: Day 13 Tbx4-LER GFP+ cells generated by directed lung mesenchymal differentiation

value: FACS sorting for single, live, GFP+ cells


title: High fat diet, Aroclor1260 input, replicate 2

value: male


title: HAEC 0.5Gy rep1

value: HAEC


title: HAEC 0Gy rep2

value: human aortic endothelial cells

value: no radiation


title: HAEC 0.1Gy rep1


title: ho-base-rep1

value: Cochlea


title: ho-apex rep1


title: High fat diet, Aroclor1260 input, replicate 1

value: Input


title: wt-middle-rep2

value: middle

value: WT


title: High fat diet, vehicle control input, replicate 5

value: Control


title: 204887840016_R06C02

type: genomic

city: Durham

first: Elizabeth

last: Martin

state: NC

address: 111 TW Alexander Drive

institute: NIEHS

description:
genomic DNA from a C3 male mouse
genomic DNA from a B6 female mouse

hyb_protocol: DNA was processed through the Infinum array protocol for hypbridization

scan_protocol: Beachip arrays were scanned on the Illumina iScan ssytem to produce Idat files

data_processing:
Data were processed using ENMix
Processed data file contains: Average Beta

label: Cy5 and Cy3

label_protocol: 500ng of DNA were bisulfite convered using the EZ gold DNA methylation kit

growth_protocol: Mice were housed from 8 to 20 weeks

extract_protocol: A 2mm cube of fresh liver tissue was homoginzed immediately following sacrifice into Buffer RLT plus with BME. DNA was then extracted using the Qiagen Allprep kit

value: M

tag: subject


title: Osteoblasts expressing homozygous WT p53

city: Houston

first: Xiaoping

last: Su

institute: MD Anderson Cancer Center

data_processing:
Basecalls were performed using bcl2fastq v2.17 for Novaseq output.
ChIP-seq reads were trimmed from 3' end until the final base had a quality score > 30, using Trimmomatic v0.33, discarding reads left with < 20 bp
ChIP-seq reads were aligned to the UCSC hg38 genome using Bowtie version 1.1.2. Only uniquely mapping reads with at most two mismatches were retained.
Peaks were called uing MACS v2.1.0 with the significance cut-off q-value <=0.05
bigWig files were generated using the bamCoverage. broadPeak files were generated using MACS v2 with default settings.
Assembly: GRCh38
Supplementary files format and content: narrowPeak

source_name: hESC-derived osteoblasts

growth_protocol: hESC clones were first cultured in StemMACS iPSC-Brew XF medium supplemented with 10 μM TGFβ inhibitor to derive MSCs which were then cultured in osteogenic differentiation medium for 24 days.

extract_protocol:
Cells were fixed with 1% formaldehyde for 10 minutes and quenched by 125mM glycine. Then the cells were sonicated in ChIP lysis buffer and then incubated with anti-p53 antibody overnight at 4°C. Magnetic beads were used to pull down the chromatin assoacated with the protein.
Library prep and sequencing were done by BGI Genomics using standard protocols

value: hESC-derived osteoblasts

tag: chip antibody

value: anti-p53 (SCB#sc126)


title: 204887840016_R04C02

description:
genomic DNA from a B6 male mouse
genomic DNA from a B6 female mouse

value: liver


title: 204887840016_R03C02

description:
genomic DNA from a B6 female mouse
genomic DNA from a B6 female mouse

value: female

value: F


title: HAEC 0Gy rep1


title: wt-middle-rep1


title: 204887840016_R01C02


title: High fat diet, vehicle control input, replicate 3


title: wt-base-rep2

value: base


title: PF3_PFD_6143

city: South San Francisco

first: Jason

middle: Anthony

last: Vander Heiden

address: 1 DNA Way

department: Biological Research AI Development

institute: Genentech

data_processing:
Sequencing reads were filtered and aligned using HTSeqGenie against the mouse genome assembly GRCh38.
Assembly: GRCh38
Supplementary files format and content: TSV count matrices

source_name: pulmonary fibrosis lung fibroblasts

extract_protocol:
Total RNA was extracted using the Qiagen RNeasy kit.
Libraries were prepared using the Illumina TruSeq Stranded Total RNA Library Prep Kit.

value: lung

tag: disease state

value: pulmonary fibrosis

value: fibroblast

value: 20% FBS plus 1mM pirfenidone


title: wt-base-rep1


title: Osteoblasts expressing heterozygous mutant p53R248W

address: 7300 Brompton St, Apt 5633


title: High fat diet, vehicle control input, replicate 1

value: male


title: 204887840016_R06C01


title: wt-apex rep2

value: Cochlea

value: apex


title: 204887840016_R05C01


title: PF3_PFD_6142


title: wt-apex rep1


title: 204887840016_R04C01


title: 204887840016_R05C02

description:
genomic DNA from a C3 female mouse
genomic DNA from a B6 female mouse


title: PF3_PFD_6141


title: Treated: R16M sample

city: Barcelona

first: Juanjo

last: Lozano

address: C/ Rosselló 153

department: Plataforma de Bioinformatica

institute: CIBEREHD

description: Treated: R16M sample

data_processing:
raw reads were analyzed for data quality using FastQC v0.11.5 (Babraham Bioinformatics) and filtered using skewer v0.2.2 (Jiang et al., 2014) for removing the low quality reads and trimming the Illumina adapter
Mapping of the reads were obtained using the STAR program against human genome (GRCm38) and the quantification of genes and transcripts were done with the RSEM program using GENCODE.vM15 database .
We use TMM method and limma-voom transformation from rounded expected counts to normalize the non biological variability
Assembly: mus musculus (GRCm38)
Supplementary files format and content: RSEM expected counts

source_name: CD45+CD11b+Ly6GlowLy6C+  cells

extract_protocol:
RNA extraction from PB monocytes was performed as previously described (Sanjurjo et al., 2018a). Briefly, 5*105 cells/well cultured in 24-well plates were incubated for 24 h in RPMI medium containing 5% FBS and the indicated stimuli. Cells were then washed with PBS and disrupted with TRIzol reagent (15596026, Thermo Fisher Scientific). Likewise, pellet from cells sorted by flow cytometry (CD45+CD11+Ly6C+Ly6Glow) were also disrupted with TRIzol reagent. For RNA extraction from mouse tumors frozen, tissue was submerged in lysis buffer with β-mercaptoethanol and disrupted mechanically using the homogenizer polytron PRO 200®. In all cases, total RNA was extracted using the RNeasy Mini Kit (74106, Qiagen).
Sequencing libraries were prepared following “TruSeq Stranded mRNA Sample Preparation Guide (Part 15031058 Rev. E)” using the “TruSeq® Stranded mRNA Library Prep” kit (20020594, Illumina Inc.) and TruSeq RNA CD Index Plate (96 Indexes, 96 Samples) (20019792, Illumina Inc.). Starting from 150 ng of total RNA, mRNA was purified, fragmented and primed for cDNA synthesis. cDNA first strand was synthesized with SuperScript-II Reverse Transcriptase (18064-014, Thermo Fisher Scientific) for 10 min at 25 °C, 15 min at 42 °C, 15 min at 70 °C and pause at 4 °C. cDNA second strand was synthesized with Illumina reagents at 16 °C for 1 hour. Then, A-tailing and adaptor ligation were performed. Finally, enrichment of libraries was achieved by PCR (30 s at 98 °C; 15 cycles of 10 sec at 98 °C, 30 s at 60 °C, 30 s at 72 °C; 5 min at 72 °C and pause at 4 °C). Afterwards, libraries were visualized on an Agilent 2100 Bioanalyzer using Agilent High Sensitivity DNA kit (5067-4626, Agilent Technologies) and quantified using Qubit dsDNA HS DNA Kit (Q32854, Thermo Fisher Scientific)

value: Treated


title: 204887840016_R03C01


title: PF3_FBS_6140

value: 20% FBS


title: Treated: R14M sample

description: Treated: R14M sample


title: 204887840016_R02C01


title: PF3_FBS_6138


title: Treated: R13M sample

description: Treated: R13M sample


title: High fat diet, vehicle control input, replicate 4

value: Control

value: Input


title: PF3_SF_6137

value: serum free DMEM medium


title: 204887840016_R01C01


title: Treated: R9M sample

description: Treated: R9M sample


title: PF3_SF_6136


title: 204887840015_R06C02

value: M


title: High fat diet, vehicle control input, replicate 2

value: liver


title: PF3_SF_6135


title: Control: R4M sample

description: Control: R4M sample


title: 204887840015_R05C02

value: female


title: 204887840015_R04C02


title: PF3_FBS_6139


title: Control: R2M sample

description: Control: R2M sample


title: PF2_PFD_6133


title: meRIP_IR-4

city: Haerbin

first: Yongliang

last: Hua

state: Heilongjiang

address: Department of Pediatric Surgery, the First Affiliated Hospital of Harbin Medical University, Key Laboratory of Hepatosplenic Surgery, Harbin, Heilongjiang, China

institute: 哈医大一院

data_processing:
RNA libraries were run on Illumina NovaSeq6000, basecalling was performed using Real-Time Analysis (RTA) v3.3.3
FastQC V0.11.7 was used for quality contriol
For MeRIP-seq, trimmed raw data performed by Trimmomatic V0.32 software, and mapped to the reference genome by using hisat2 (v2.1.0). The m6A peaks were identified using exomePeak R package (v2.13.2).
For RNA-seq, rawdata were trimmed by cutadapt software, and mapped to genome by Hisat2, differentially expressed genes analyzed by Ballgown R package
Genome_build: mm10

extract_protocol:
The purity and concentration of total RNA samples were determoned withNanoDrop ND-1000. Intact mRNA was isolated from total RNA samples using Arraystar Seq-Star ploy(A) mRNAIsolation Kit according to the manufacturer's protocol.Isolated mRNA was chemcally fragmented into 100-nucleotide-long fragments by incubation in fragmentation buffer.
NEBNext® Poly(A) mRNA Magnetic Isolation Module (New England Biolabs) RiboZero Magnetic Gold Kit (Human/Mouse/Rat) (Epicentre, an Illumina Company) KAPA Stranded RNA-Seq Library Prep Kit (Illumina)

treatment_protocol: The liver samples were immediately collected into RNase-free centrifuge tubes with 1 ml RNA later (Ambion) in accordance with the manufacturer’s instructions, left overnight at 4 °C and stored at − 80 °C to prevent RNA isolation

tag: molecule subtype

value: MeRIP-RNA


title: PF2_PFD_6132

tag: tissue


title: 204887840015_R01C02

tag: Sex

value: F


title: PF2_FBS_6130

value: lung


title: Control: R3M sample

description: Control: R3M sample

tag: group


title: PF2_FBS_6131

value: pulmonary fibrosis


title: 204887840015_R05C01


title: PF2_FBS_6129

value: fibroblast


title: PF2_PFD_6134

value: 20% FBS plus 1mM pirfenidone


title: 204887840015_R02C02


title: PF2_SF_6128


title: 204887840015_R04C01

value: male


title: meRIP_IR-5

tag: age


title: 204887840015_R03C02


title: meRIP_IR-2


title: PF2_SF_6127


title: 204887840015_R03C01


title: meRIP_IR-3


title: meRIP_Sham-4


title: 204887840015_R02C01


title: PF2_SF_6126


title: meRIP_Sham-3


title: 204887840015_R01C01


title: N1_PFD_6125

source_name: normal human lung fibroblasts

value: normal


title: 204887840013_R06C02


title: meRIP_Sham-2

source_name: liver


title: N1_PFD_6124


title: meRIP_Sham-1


title: N1_PFD_6123


title: 204887840013_R05C02


title: N1_FBS_6122


title: polyA_IR-5

value: mRNA


title: meRIP_Sham-5


title: polyA_IR-4


title: 204887840013_R04C02


title: 204887840013_R03C02


title: N1_FBS_6121


title: 204887840013_R02C02


title: 204887840013_R01C02


title: polyA_IR-3


title: polyA_IR-2


title: N1_FBS_6120


title: N1_SF_6118


title: 204887840013_R06C01


title: N1_SF_6119


title: 204887840013_R05C01


title: N1_SF_6117


title: polyA_Sham-4


title: OV14

city: Beijing

first: Xuncheng

last: Wang

address: 5 Yiheyuan Road

institute: Peiking University

description: polyA RNA isolated

data_processing:
Clean reads were filtered by removing reads containing ploy-N, reads containing adapter, and low-quality reads from the raw data. The filtered clean reads were aligned to the N. benthamiana reference genome using Hisat2 v2.0.4 with default parameters. The expression level for each transcript was normalized and estimated using the fragments per kilobases per million reads (FPKM) method (Trapnell et al., 2010).
Genome_build: Niben101
Supplementary_files_format_and_content: tab-delimited text files include FPKM values for each Sample.

organism: Nicotiana benthamiana

source_name: seedlings

growth_protocol: Nicotiana benthamiana seeds were surface sterilized with 70% (w/v) ethanol for 30 s and 5% (v/v) H2O2 for 30 min, then washed with sterile H2O and planted on Murashige and Skoog (MS) medium for germination under a 16 h light/8 h dark cycle at 26°C in a growth chamber. The seedlings of N. benthamiana were transferred to the soil mix and grown under greenhouse conditions.

extract_protocol:
Total RNA was isolated from N. benthamiana plant tissue using a TransZol Plant RNA Kit (TransGen Biotech, Beijing, China).
The cDNA libraries were generated using NEBNext? Ultra? RNA Library Prep Kit for (Illumina, USA) according to manufacturer's recommendations.

value: seedlings


title: polyA_Sham-3


title: 204887840013_R03C01


title: OV10

description: polyA RNA isolated


title: polyA_Sham-2


title: OV3

description: polyA RNA isolated


title: polyA_Sham-1


title: CK

description: polyA RNA isolated

value: wild type


title: 204887840013_R01C01


title: polyA_IR-1


title: polyA_Sham-5


title: meRIP_IR-1


title: 204887840013_R04C01


title: 204887840013_R02C01


title: WL_MAD.replicate_10.RNAseq

status: Public on May 11 2023

city: Chengdu

first: jin

last: long

address: Hui ming

institute: Sichuan Agricultural University

description:
whole.Pig.PCG.TPM.xls
whole.Pig.TUCP.TPM.xls
whole.Pig.lncRNA.TPM.xls
whole.Pig.PCG.counts.xls
whole.Pig.TUCP.counts.xls
whole.Pig.lncRNA.counts.xls

data_processing:
Gene-level expression was estimated as transcripts per million (TPM) using the high-speed transcript quantification tool Kallisto (version 0.43.0).
Tpm and counts matrixs were generated using R vresion 3.6.1.
AssemblySusscrofa11.1
Matrix table with raw gene counts for every gene and every sample

organism: Sus scrofa

source_name: mesenteric adipose

extract_protocol:
Total RNA was extracted using the RNeasy Mini Kit (Qiagen).
We used an rRNA depletion protocol (Ribo-Zero kit, Epicentre) coupled with the Illumina TruSeq stranded RNA-Seq library protocol to construct the RNA-Seq libraries. All libraries were quantified using the Qubit dsDNA High Sensitivity Assay Kit (Invitrogen) and sequenced on HiSeq X Ten (Illumina) platform to produce average ~ 11.97 Gb high-quality data for each library.

value: mesenteric adipose


title: WL_MAD.replicate_3.RNAseq


title: WL_ULB.replicate_7.RNAseq

source_name: upper layer of backfat

value: upper layer of backfat


title: WL_MAD.replicate_4.RNAseq


title: WL_MAD.replicate_1.RNAseq


title: WL_ULB.replicate_9.RNAseq


title: WL_MAD.replicate_9.RNAseq


title: WL_MAD.replicate_8.RNAseq


title: WL_ULB.replicate_1.RNAseq


title: WL_MAD.replicate_7.RNAseq


title: WL_ULB.replicate_3.RNAseq


title: WL_ULB.replicate_2.RNAseq


title: WL_MAD.replicate_6.RNAseq


title: WL_MAD.replicate_2.RNAseq


title: WL_ULB.replicate_6.RNAseq


title: WL_RAD.replicate_9.RNAseq

source_name: retroperitoneal adipose

value: retroperitoneal adipose


title: WL_ULB.replicate_10.RNAseq


title: WL_ULB.replicate_5.RNAseq


title: WL_ULB.replicate_4.RNAseq


title: WL_RAD.replicate_10.RNAseq


title: WL_RAD.replicate_7.RNAseq


title: WL_RAD.replicate_8.RNAseq


title: WL_ULB.replicate_8.RNAseq


title: WL_RAD.replicate_5.RNAseq


title: WL_RAD.replicate_4.RNAseq


title: WL_RAD.replicate_3.RNAseq


title: WL_MAD.replicate_5.RNAseq


title: WL_RAD.replicate_6.RNAseq


title: WL_RAD.replicate_2.RNAseq


title: WL_RAD.replicate_1.RNAseq


title: WL_GOM.replicate_8.RNAseq

source_name: greater omentum fat

value: greater omentum fat


title: WL_GOM.replicate_3.RNAseq


title: WL_GOM.replicate_2.RNAseq


title: WL_GOM.replicate_6.RNAseq


title: WL_GOM.replicate_5.RNAseq


title: WG_ULB.replicate_46.RNAseq


title: WL_GOM.replicate_7.RNAseq


title: WL_GOM.replicate_9.RNAseq


title: WG_ULB.replicate_45.RNAseq


title: WL_GOM.replicate_4.RNAseq


title: B16_CD8_Tumor_PD1nega_CD4PDL1_sup1

city: Noda

first: Hiroyasu

last: Aoki

state: Chiba

address: 2669 Yamazaki

department: Research Institute for Biomedical Sciences

country: Japan

institute: Tokyo University of Science

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 by using Cutadapt-3.2  (Martin et al., 2011) and PRINSEQ-0.20.4 (Schmieder et al., 2011). Sequencing data were processed by MiXCR-3.0.5 (Bolotin et al., 2015). In MiXCR, filtered reads were aligned to reference mouse TCR V/D/J sequences registered in the international ImMunoGeneTics (IMGT) information system: -vParameters.geneFeatureToAlign = VTranscript -vjAlignmentOrder = JThenV, then identical sequences were assembled and grouped in clones with PCR and sequencing error correlation with the following parameters: -badQualityThreshold=10, –separateByV=true, --only-productive=true, –region-of-interest=CDR3. The Variable (V) and Joining (J) segment of TCRs were represented in IMGT gene nomenclature.
List of final clones were analyzed by VDJtools-1.2.1 (Shugay et al., 2015). Then, the sequencing reads of sample was normalized to the cell count in each sample by “DownSample” command of VDJtools. T-cell clones were determined as TCR reads with the same TCR V segment, J segment and CDR3 nucleotide sequence.
Assembly: IMGT reference sequences of mouse 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

source_name: tumor on day 14 after innoculation of LLC

extract_protocol:
Intravascular leukocytes were stained by i.v. injection of FITC-conjugated anti-CD45.2 mAb (3 mg/mouse, clone 104, BioLegend) three minutes before sacrifice.
dLN cells were prepared by standard method. Tumors were cut into small fragments and digested for 45 minutes at 37°C with 0.1% collagenase (FUJIFILM Wako), then subjected to 40% Percoll PLUS (Cytiva) and Histopaque-1083 (Sigma-Aldrich) gradient, and leukocytes were recovered from the interphase. The cell concentration of the suspensions was determined using Flow-Count fluorospheres (Beckman Coulter) and a Cytoflex flow cytometer (Beckman Coulter).
Cells were stained with a mix of Fc Block (anti-mouse CD16/CD32 mAb; clone 93, BioLegend) and fluorophore-conjugated anti-mouse mAbs. For dLN samples, Lineage+ (CD11b, B220, NK1.1, Ter119) cells were depleted using BD IMag Streptavidin Particles Plus-DM (BD Biosciences). For tumor samples, TCRb+ T cells were enriched magnetically using Anti-APC MicroBeads (Miltenyi) and LS columns (Miltenyi).
CD8+, CD8+ PD1-, CD8+ PD1+ Ly108+ Tim3-, CD8+ PD1+ Ly108+ Tim3+, and CD8+ PD1+ Ly108- Tim3+ T cells from tumor, and CD8+ CD44high T cells from dLN were sorted using FACS Aria II or Aria III (BD Biosciences). Nonviable cells were excluded from the analysis based on forward and side scatter profiles and propidium iodide staining. Intravascular leukocytes were also excluded. Purity of sorted cells was always over 95%.
Bulk TCR sequencing library was prepared according to the previous report (Aoki et al., 2021). In brief, to amplify the TCR cDNA containing complementarity determining region 3 (CDR3), nested PCR of the TCR locus was performed as follows. Beads containing cDNA was resuspended with the 12.5 mL of first PCR mixture comprised 0.5 mL of 10 mM trP1 primer, and 1 mL of TCR primer mix (Trac_ex, and Trbc_ex), 4.75 mL of water, and 6.25 mL of KAPA Hifi Hotstart ReadyMix (KAPA Biosystems). The thermal cycling conditions were programmed as follows: denaturation at 95°C for 3 minutes, 5 cycles of denaturation for 20 seconds at 98°C, annealing for 15 seconds at 65°C and extension for 30 seconds at 72°C, followed by a final extension at 72°C for 2 minutes. Next, 2.5 mL of the first-PCR products were added into the 22.5 mL of second PCR mixture consisted of 1.75 mL of 10 mM primer mix (trP1, Trac_in-Bio, and Trbc_in-Bio), 8.25 mL of water and 12.5 mL of KAPA Hifi Hotstart ReadyMix. The thermal cycling conditions were the same as the first PCR except for the cycle number, which was 25 cycles. Next, 25 mL of the second-PCR products were purified using Agencort AM Pure XP kit (Bexkman-Coulter) at a 0.7:1 ratio (beads to sample) and eluted in 20 mL of 10mM of Tris HCl pH 8.0. Fragmentasion and adaptor ligation were performed using 10-20ng of second-PCR product as a template with NEBNext FS DNA Library Prep Kit (New England Biolabs) with some modifications: NEBNext Adaptor for Illumina in “Adaptor Ligation” step was substituted by 5mM Adaptor P1, and the reaction volume was 1/4 of the recommended in all steps. Adaptor-ligated DNA was purified using Agencort AM Pure XP kit (Beckman Coulter) at a 0.8:1 ratio (beads to sample) and eluted in 20 mL of 10mM of Tris HCl pH 8.0. The third PCR was carried out using barcoded primers to enrich the TCR cDNA flanked with sequencing adapters. The third PCR mixture consisted of 1 mL of 10 mM trP1 primer, 1 mL of 10 mM IonA-BC-Trbc primer, 3 mL of template and 5 mL of NEBNext Ultra II Q5 Master Mix (accessory of NEBNext FS DNA Library Prep Kit). The thermal cycling conditions were programmed as follows: denaturation at 98°C for 30 seconds, 10 cycles of denaturation for 10 seconds at 98°C, annealing and extension for 1minute 15 seconds at 65°C, followed by a final extension at 65°C for 5 minutes. The PCR product was purified and subjected to size selection using Agencort AM Pure XP kit (Beckman Coulter) at a 1.0:1 ratio (beads to sample) and eluted with 20 mL of Tris-HCl (pH 8.0). Amplified TCR libraries were quantified using a KAPA SYBR Fast qPCR Kit (KAPA Biosystems) and size distribution was analyzed by Microchip Electrophoresis System MultiNA (Shimadzu, Kyoto, Japan). Primers used for library preparations were summarized in Supplementary Table 1.  Final TCR libraries, whose lengths were 200–300 base pairs, were pooled and sequenced using an Ion 540 Kit Chef, an Ion 540 Chip kit, and an Ion Genestudio S5 Sequencer (Thermo Fisher Scientific) according to the manufacturer’s instructions, except the flow number (500).

treatment_protocol: B16F10 or LLC cells (5 × 105 cells except for sample 103-107: 2 × 105 cells) were inoculated subcutaneously (s.c.) into the right flanks of C57BL/6 mice. Anti-PD-L1 mAb (clone 10F.9G2, BioLegend) or Anti-CD4 mAb (clone GK1.5, BioLegend) was injected intraperitoneally (i.p.) at a dose of 200 μg per mouse on days 5 and 9 after tumor inoculation.

value: combination of anti-CD4 and anti-PD-L1 mAbs

value: Intravenous (i.v.) staining-negative, lineage-negative (CD11b, B220, NK1.1 and Ter119), TCRbeta+ and CD4- CD8+ T cells

value: 8 weeks

tag: tumour cell line

value: B16F10 melanoma

tag: organ

tag: date


title: B16_CD8_Tumor_CD4PDL1_sup1

tag: treatment

tag: cell type

value: female


title: B16_CD8_Tumor_Tim3_CD4PDL1_sup1

tag: strain


title: B16_CD8_Tumor_DP_CD4PDL1_sup1


title: LLC_CD8_Tumor_PDL1_5

value: anti-PD-L1 mAb


title: B16_CD8_Tumor_Ly108_CD4PDL1_sup1