title: SOX8-specific siRNA, rep 3

city: Singapore

first: Reshma

last: Taneja

address: National University of Singapore Dept of Physiology, Blk MD9 Level 2, 2 Medical Drive,

department: Physiology

country: Singapore

institute: National University of Singapore

description:
siSOX8_3
KD_S83
SiScr_siSox8_read_counts.xlsx

data_processing:
Raw image data file from sequencing was transformed to Sequenced Reads by CASAVA base recognition (Base Calling).
Bcl2fastq was used for basecalling.
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence, then mapped to hg19 whole genome using STAR v2.5 with parameters mismatch=2. STAR used the method of Maximal Mappable Prefix (MMP) which can generate a precise mapping result for junction reads.
HTSeq v0.6.1 was used to count the read numbers mapped of each gene. FPKM of each gene was calculated based on the length of the gene and reads count mapped to this gene. FPKM, Reads Per Kilobase of exon model per Million mapped reads, considers the effect of sequencing depth and gene length for the reads count at the same time, and is currently the most commonly used method for estimating gene expression levels.
Differential expression analysis between two condition/groups (three biological replicates per condition) was performed using the DESeq2 R package (v2_1.6.3). DESeq2 provide statistical routines for determining differential expression in digital gene expression data using a model based on the negative binomial distribution. The resulting P-values were adjusted using the Benjamini and Hochberg’s approach for controlling the False Discovery Rate(FDR). Genes with an adjusted P-value <0.05 found by DESeq2 were assigned as differentially expressed.
Assembly: hg19
Supplementary files format and content: excel file (.xlsx) containing matrix with raw counts and normalised counts (FPKM) for all samples

source_name: RD

growth_protocol: RD cells were cultured in RPMI media supplemented with 10% fetal bovine serum and 1% Penicillin-Streptomycin

extract_protocol:
Total RNA extraction with Trizol. RNA purity and integrity were assessed with Nanodrop and agarose gel electrophoresis. mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. The mRNA was first fragmented randomly by addition of fragmentation buffer. The first strand of cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (RNase H-).
RNA libraries were prepared for sequencing using NEBNext Ultra RNA Library Prep Kit for Illumina

treatment_protocol: Control(siscr) and SOX88-knockdown (siSOX8) cells were transfected with scrambled siRNA and SOX8-specific siRNA respectively using Lipofectamine RNAiMAX.

value: RD

value: embryonal rhabdomyosarcoma (ERMS)

value: FN-RMS

value: SOX8-specific siRNA


title: Cancer patient 16 (Validation cohort 1)

status: Public on May 16 2025

type: SRA

city: Chicago

first: Cheng-Wei

last: Ju

state: Illinois

address: 929 East 57th Street, GCIS, Room E313

country: USA

institute: The University of Chicago

description: Library name: NL-09

library_source: other

data_processing:
Sequence reads were trimmed for adaptor sequence/low-quality sequence using Cutadapt (4.6)
PCR duplicates were removed with the BBMap tool (https://sourceforge.net/projects/bbmap/).
Random barcodes at reads end were trimmed, and low-quality reads were removed using the cutadapt tool again
Then the remaining reads were aligned to the human genome (hg38) using  bowtie2 (version 2.5.1) allowing a maximum of three mismatches.
Read count extraction and differential expression levels of exons and genes were analyzed using RADAR (0.2.4) followed by DEseq2 (1.36.0).
Cleaned reads that failed to align to the human genome were applied using Kraken2 with its standard database, which contains bacterial, archaeal, viral, and human sequences. Based on the aligned results from Kraken2, we normalized the data by calculating the reads per million (RPM). Then we performed student t-test (two tailed) to calculate the p value for the differential expression.
We identified microbial species in cfRNA samples using kraken2 (version 2.1.3)52, setting a threshold whereby the median reads of the specie must exceed 10 in at least one group (CRC or non-cancer). Following species identification, we extracted their sequences from the kraken2 reference genome. To construct a new, streamlined reference, we limited each microbial species to a maximum of ten reference genomes, minimizing redundancy Subsequently, we aligned the reads that failed to align to the human genome to this curated microbiome reference genome using bowtie2. The alignments generated by bowtie2 were applied in the calculation of the microbiome mapping ratio. Variant identification within these sequences is then achieved through the precise analysis of base composition at each genomic position, employing bam-readcount tool (https://github.com/genome/bam-readcount). The generated bam-readcount output results were parsed and analyzed to calculate the misincorporation ratio.
Assembly: hg38 (GRCh38.107)
Assembly: Kraken 2 standard database
Supplementary files format and content: abundance.host.csv|containing the abundance (raw reads), fold change, p value of various human transcirpts from cfRNA.
Supplementary files format and content: abundance.microbiome.csv|containing the RPM, fold change, p value of various microbial abundance from cfRNA.
Supplementary files format and content: mutation.microbiome.csv |containing the p value, fold change, and mutation ratio at modified sites in microbiome-dervied cfRNA.
Supplementary files format and content: mutation.host.rRNA.csv |containing the p value, fold change, and mutation ratio at human rRNA from cfRNA.
Supplementary files format and content: mutation.host.tRNA.csv |containing the p value, fold change, and mutation ratio at human tRNA from cfRNA.
Supplementary files format and content: mutation.hepG2.csv |containing mutation ratio of human tRNA from HepG2.

molecule: other

organism: Homo sapiens

source_name: plasma

extract_protocol:
cfRNAs were isolated from 600 μl plasma using QIAamp ccfDNA/RNA Kit (Qiagen, Cat. 55184) according to the manufacturer's protocol. In brief, 180 μl RPL buffer was added into 600 μl plasma, followed by vortexing for 5 s. After leaving at room temperature for 3 mins, 100 μl RPP buffer is added. Then close EP tube cap and immediately mix vigorously by vortexing 10 s for 2 times, which is important to disperse the solid. Incubate the EP tube on ice for 3 min. After the standard steps of washing and concentration following the manufacturer's protocol, the purified cfRNA was finally eluted in 14 µl of RNase-free water.
Plasma cell-free RNA (cfRNA) or small RNA from HepG2 (<200 nt) were fragmented to lengths of ~35-55 nucleotides using the 10X RNA Fragmentation Reagent (AM8740, Invitrogen), which was used as 15X with heating at 70 °C for 14 mins. Purify the fragmented RNA using the Oligo Clean & Concentrator kit (Zymo Research) with a final elution volume of 10 μl. The subsequent 3′-end repair involved the preparation of a mixture of fragmented RNA, 2 μl of 10× T4 Polynucleotide Kinase Reaction Buffer (B0201S, NEB), 2 μl of T4 PNK, 1 μl SUPERase•In RNase Inhibitor and a proper volume of RNase-free water. Mix the 20 μl reaction mixture very well and incubate at 37°C for 1 hour. 3’-end-repaired RNA fragments were purified by Oligo Clean & Concentrator kit and eluted in 10 μl of RNase-free water. For 3′-adapter ligation, RNA fragments were denatured with 1ul of 10 μM RNA 3′-linker (5′rApp-NNNNN AGATCGGAAGAGCGTCGTG-3SpC3) by heating at 70°C for 2 minutes and then immediately moved onto ice. Then a mixture of 2.5 μl of 10× T4 RNA Ligase Reaction Buffer (NEB), 7.5 μl of 50% PEG8000, 1 μl SUPERase•In, and 2 μl T4 RNA ligase 2 truncated KQ (NEB) was prepared, followed by mixing well and incubating at 25°C for 2 hrs and then at 16°C for 10 hrs. The excessive adapters were removed by adding 2 μl of 5′-deadenylase (NEB) with an incubation at 30 °C for 45 mins, followed by adding 1.0 μl of RecJf (NEB) with an incubation at 37 °C for 45 mins. The 3’-ligated RNA was then purified by RNA Clean & Concentrator (Zymo Research) and eluted in 10 μl of RNase-free water. Reverse transcription was initiated by mixing the 3’-ligated RNA with 1 ul of 2.0 μM RT primer (5′-ACACGACGCTCTTCCGATCT-3′) and heating at 65°C for 2 minutes. Then move the samples onto ice immediately. A mixture of 2 μl of 10× AMV Reverse Transcriptase Reaction Buffer (NEB), 2 μl of 10 mM dNTPs, 1 μl RNaseOUT (Thermo Fisher Scientific), 2 μl HIV Recombinant Reverse Transcriptase (Worthington) and a proper volume of RNase-free water was prepared, to give a final volume of 20 μl for a one-hour incubation at 37°C. Upon the completion of RT, 1.0 μl volume of RNase H (NEB, M0297L) was added to the reaction mixture, followed by incubation at 37 °C for 20 min and 70 °C for 5 min. The produced cDNAs after RT were purified by Oligo Clean & Concentrator (Zymo Research) and eluted in 10 μl of RNase-free water. A mixture of eluted cDNA and 2.0 μl of 10 μM cDNA 3′-linker (5′Phos-NNNNNAGATCGGAAGAGCACACGTCTG-3SpC3) was denatured at 75 °C for 2 min and then moved onto ice immediately. 3-μl of 10× T4 RNA Ligase Reaction Buffer (NEB), 15 μl of 50% PEG8000, 3 μl of 10 mM ATP, and 1.0 μl of T4 RNA ligase 1 (high concentration, NEB) were added to the cDNA–adapter mixture and mixed very well. Incubate the ligation reaction mixture at 25 °C for 12 h. The libraries were amplified using universal and indexed primers from NEBNext Multiplex Oligos for Illumina (NEB). All libraries were sequenced on an Illumina NovaSeq 6000 or NovaSeqX with SE100 bp mode. The LIME-seq method enables high mutation ratio detection across multiple RNA modifications, including m1A, m3C, m1G, m22G, m3U, etc., with an enhanced read-through at these methylation sites. Such performance has been facilitated by the robust HIV RT enzyme, and it effectively uncovered methylation fraction information at methylated sites

tag: tissue

value: plasma

tag: genotype

value: Cancer patient


title: Murine CAFs treated with 0.2 mM glutamine + DMSO, Rep2

city: La Jolla

first: Cosimo

last: Commisso

state: CA

address: 10901 N. Torrey Pines Road

department: Cancer Metabolism and Microenvironment Program

institute: Sanford Burnham Prebys Medical Discovery Institute

description: Library name: 02Qveh02

library_source: transcriptomic

data_processing:
Bulk RNA-seq samples were processed using nf-core rnaseq pipeline v3.14.090 using parameters “--aligner star_rsem” to perform read trimming, quality control, alignment, gene quantification and normalization. We used mouse mm10 genome sequence and gene annotations provided by 10X Genomics (refdata-gex-mm10-2020-A) with the nf-core pipeline. Differential expression and gene set enrichment analysis (GSEA) were performed using nf-core differential abundance pipeline v1.5.0 with parameters ‘--filtering_min_abundance 4 --filtering_min_proportion 0.50 --differential_min_fold_change 1.5 --gsea_run true --gsea_permute "gene_set" --gsea_nperm 1000’ and mouse gene sets v2023.2.Mm from MSigDB.
Assembly: mm10
Supplementary files format and content: Tab-delimited Transcripts Per Million (TPM)

molecule: total RNA

organism: Mus musculus

source_name: Immortalized primary CAFs

extract_protocol:
Total RNA was extracted using a PureLink RNA Mini Kit (Invitrogen).
PolyA RNA isolation and library preparation was performed with the Watchmaker mRNA Library Prep Kit (Watchmaker Genomics: 7BK0001) with xGEN Stubby adaptors (IDT, Cat# 336338420) and xGEN 10nt UDIs (IDT, Cat# 336338436). Libraries were sequenced (2x75bp) with the Element Biosciences AVITI Sequencing platform using the AVITI 2x75 High Output Cloudbreak Freestyle Kit (Element Biosciences, Cat# 860-00015).

treatment_protocol: Murine CAFs were treated with or without 25 mM EIPA in 0.2 mM glutamine, or treated with or without 25 pg/mL IL-1a in 4 mM glutamine, for 32 hours. DMSO was used as the vehicle for EIPA and added to each treatment to maintain consistency across all conditions.

tag: cell type

value: Immortalized primary CAFs

tag: treatment

value: 0.2 mM glutamine + DMSO


title: SOX8-specific siRNA, rep 2

description:
siSOX8_2
KD_S82
SiScr_siSox8_read_counts.xlsx

tag: cell line


title: SOX8-specific siRNA, rep 1

description:
siSOX8_1
KD_S81
SiScr_siSox8_read_counts.xlsx


title: Murine CAFs treated with 0.2 mM glutamine + DMSO, Rep1

description: Library name: 02Qveh01


title: scrambled siRNA, rep 3

description:
siscr_3
WT_S83
SiScr_siSox8_read_counts.xlsx

value: scrambled siRNA


title: Cancer patient 15 (Validation cohort 1)

description: Library name: NK-11


title: scrambled siRNA, rep 2

description:
siscr_2
WT_S82
SiScr_siSox8_read_counts.xlsx


title: Murine CAFs treated with 0.2 mM glutamine + 25 uM EIPA, Rep5

description: Library name: 02Qeipa05

value: 0.2 mM glutamine + 25 uM EIPA


title: Cancer patient 14 (Validation cohort 1)

description: Library name: NJ-12


title: Murine CAFs treated with 0.2 mM glutamine + 25 uM EIPA, Rep4

description: Library name: 02Qeipa04


title: Cancer patient 13 (Validation cohort 1)

description: Library name: NJ-10


title: EHMT1-specific siRNA, replicate 3

data_processing:
Raw image data file from sequencing was transformed to Sequenced Reads by CASAVA base recognition (Base Calling).
Bcl2fastq was used for basecalling.
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence, then mapped to hg19 whole genome using STAR v2.5 with parameters mismatch=2. STAR used the method of Maximal Mappable Prefix (MMP) which can generate a precise mapping result for junction reads.
HTSeq v0.6.1 was used to count the read numbers mapped of each gene. FPKM of each gene was calculated based on the length of the gene and reads count mapped to this gene. FPKM, Reads Per Kilobase of exon model per Million mapped reads, considers the effect of sequencing depth and gene length for the reads count at the same time, and is currently the most commonly used method for estimating gene expression levels.
Differential expression analysis between two condition/groups (three biological replicates per condition) was performed using the DESeq2 R package (v2_1.6.3). DESeq2 provide statistical routines for determining differential expression in digital gene expression data using a model based on the negative binomial distribution. The resulting P-values were adjusted using the Benjamini and Hochberg’s approach for controlling the False Discovery Rate (FDR). Genes with an adjusted P-value <0.05 found by DESeq2 were assigned as differentially expressed.
Assembly: hg19
Supplementary files format and content: tab text file includes raw counts for each sample

source_name: Cell line

treatment_protocol: Control (siscr) and EHMT1-knockdown (siEHMT1) cells were transfected with scrambled siRNA and EHMT1-specific siRNA respectively using Lipofectamine RNAiMAX.

value: Cell line

value: ERMS

value: EHMT1-specific siRNA


title: EHMT1-specific siRNA, replicate 1


title: EHMT1-specific siRNA, replicate 2


title: scrambled siRNA, replicate 3


title: Cancer patient 12 (Validation cohort 1)

description: Library name: NI-07


title: scrambled siRNA, rep 1

description:
siscr_1
WT_S81
SiScr_siSox8_read_counts.xlsx


title: scrambled siRNA, replicate 2


title: Murine CAFs treated with 0.2 mM glutamine + 25 uM EIPA, Rep2

description: Library name: 02Qeipa02


title: Murine CAFs treated with 0.2 mM glutamine + 25 uM EIPA, Rep3

description: Library name: 02Qeipa03


title: Cancer patient 18 (Validation cohort 1)

description: Library name: NL-11


title: scrambled siRNA, replicate 1


title: Cancer patient 11 (Validation cohort 1)

description: Library name: NK-10


title: Cancer patient 10 (Validation cohort 1)

description: Library name: NJ-15


title: Murine CAFs treated with 0.2 mM glutamine + 25 uM EIPA, Rep1

description: Library name: 02Qeipa01


title: rat mesentery artery_male_knockout_4

city: Tucson

first: Yong

last: Liu

state: AZ

address: The University of Arizona

institute: University of Arizona

description: Library name: rMES15LR

data_processing:
Raw RNA-Seq sequence reads were pre-processed using Trim Galore (v0.6.2) (http://www.bioinformatics.babraham.ac.uk/projects/trim_galore/). Adapters and reads with low quality (base quality < 20) were removed prior to further analysis.
Reads longer than 20bp after the trimming were aligned to the rat genome (rn7) using hisat2 (v2.2.1).
Transcript assembly and quantification were performed using StringTie2 (v2.2.1)
DESeq2 package (1.46.0) was used to perform differential gene expression analysis.
Assembly: rn7
Supplementary files format and content: FPKM file include FPKM for each sample

organism: Rattus norvegicus

source_name: mesentery artery

extract_protocol:
Total RNA was extracted from cell samples using the TRIzol reagent
RNA-seq libraries were then prepared following the whole transcriptome sequencing protocol of Novogene (https://www.novogene.com/us-en/resources/blog/a-basic-guide-to-rna-sequencing/).

value: mesentery artery

value: KO


title: rat mesentery artery_male_knockout_2

description: Library name: rMES13LR


title: rat mesentery artery_male_knockout_3

description: Library name: rMES14LR


title: rat mesentery artery_male_knockout_1

description: Library name: rMES12LR


title: Cancer patient 9 (Validation cohort 1)

description: Library name: NJ-14


title: Cancer patient 8 (Validation cohort 1)

description: Library name: NL-08


title: rat mesentery artery_male_wildtype_3

description: Library name: rMES3LR

value: WT


title: rat mesentery artery_male_wildtype_4

description: Library name: rMES4LR


title: rat mesentery artery_male_wildtype_2

description: Library name: rMES2LR


title: rat mesentery artery_male_wildtype_1

description: Library name: rMES1LR


title: male 42 sperm, fresh

city: Auburn University

first: Xu

last: Wang

state: AL

address: 273 CASIC Building, Auburn Research Park, 559 Devall Dr

department: Pathobiology, College of Veterinary Medicine

institute: Auburn University

description: Library name: BB_42_Fresh1

data_processing:
The raw reads quality was assessed by FastQC (version 0.11.6)
Low-quality bases and adapter sequences were trimmed using Trimmomatic (version 0.39)
RNA-seq reads were aligned to the blue catfish (Ictalurus furcatus) reference genome using TopHat (version 2.1.1)
The gene read counts of each sample were quantified and summarized using BEDTools.
Assembly: GCA_023701845.2
Supplementary files format and content: CSV file includes gene read counts for each sample

molecule: polyA RNA

organism: Ictalurus furcatus

source_name: sperm

extract_protocol:
RNA extraction wsa conducted using Qiagen AllPrep DNA/RNA Kit (Qiagen, CA).
The library for each sperm sample was constructed using the Zymo-Seq RiboFree Total RNA Library Kit (Zymo Research, Irvine, CA, U.S.)

treatment_protocol: The sperm from each male was divided into two groups. One was directly stored in -80 ℃ freezer as fresh group, the other one was cryopreserved using optimized cryopreserved protocol.

value: sperm

value: fresh


title: Cancer patient 7 (Validation cohort 1)

description: Library name: NL-07


title: male 43 sperm, fresh

description: Library name: BB_43_Fresh1


title: rat kidney cortex_male_knockout_4

description: Library name: rKC30LR

source_name: kidney cortex

value: kidney cortex


title: Cancer patient 6 (Validation cohort 1)

description: Library name: NL-06


title: male 41 sperm, fresh

description: Library name: BB_41_Fresh1


title: rat kidney cortex_male_knockout_2

description: Library name: rKC28LR


title: rat kidney cortex_male_knockout_3

description: Library name: rKC29LR


title: male 39 sperm, fresh

description: Library name: BB_39_Fresh1


title: rat kidney cortex_male_knockout_1

description: Library name: rKC27LR


title: rat kidney cortex_male_wildtype_3

description: Library name: rKC19LR


title: Cancer patient 5 (Validation cohort 1)

description: Library name: NL-05


title: male 37 sperm, fresh

description: Library name: BB_37_Fresh1


title: rat kidney cortex_male_wildtype_2

description: Library name: rKC18LR


title: rat kidney cortex_male_wildtype_1

description: Library name: rKC16LR


title: Cancer patient 4 (Validation cohort 1)

description: Library name: NL-19


title: Cancer patient 3 (Validation cohort 1)

description: Library name: NL-18


title: Lungs, 200 mg/mL silica, Day28, bio rep 6

city: Beijing

first: Jiaqi

last: Lv

address: Nanwei Road, Xicheng District

country: China

institute: Chinese Center for Disease Control and Prevention

data_processing:
adaptor sequence was removed by Cutadapt v 4.0
Sequence reads were trimmed for low-quality sequence using FastQC Version 0.11.9  (parameter- Quality limit: Q20)
Trimmed sequence reads were mapped to Rnor_6.0 using HISAT2 v2.1.0 with default parameters.
Read count extraction and normalization were performed using HTSeq (0.9.1)
Assembly: rn6
Supplementary files format and content: tab-delimited text files include FPKM values for each Sample

source_name: lung

growth_protocol: Specific pathogen free Wistar rats (5-6 w, 170-190 g, male)  were given free access to standard diet and water and were housed with temperature (18-25 ℃) and humidity (40-70%) control and with a light-dark cycle of 12 h/12 h.

extract_protocol:
total RNA of lung tissues of rats exposed to 200 mg/mL silica particles and NS for 14 d and 28 d was extracted using Trizol reagent (15596018, Invitrogen, Thermo Fisher Scientific, Inc.).
Sequencing libraries were generated according to the following steps. Firstly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in an Illumina proprietary fragmentation buffer. First strand cDNA was synthesized using random oligonucleotides and Super Script II. Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities and the enzymes were removed. After adenylation of the 3′ ends of the DNA fragments, Illumina PE adapter oligonucleotides were ligated to prepare for hybridization. To select cDNA fragments of the preferred 400-500 bp in length, the library fragments were purified using the AMPure XP system (Beckman Coulter,Beverly, CA, USA).

treatment_protocol: All the rats were administered with silica particles or normal saline via intratracheal instillation.

tag: strain

value: Wistar

value: lung

value: 200 mg/mL silica

tag: time

value: Day 28

tag: Sex

value: male


title: Lungs, 200 mg/mL silica, Day28, bio rep 5


title: Lungs, 200 mg/mL silica, Day28, bio rep 4


title: male 44 sperm, fresh

description: Library name: BB_44_Fresh1


title: male 35 sperm, fresh

description: Library name: BB_35_Fresh1


title: Lungs, 200 mg/mL silica, Day28, bio rep 3


title: Cancer patient 1 (Validation cohort 1)

description: Library name: NI-05

value: plasma

value: Cancer patient


title: Lungs, 200 mg/mL silica, Day28, bio rep 2


title: male 34 sperm, fresh

description: Library name: BB_34_Fresh1


title: Lungs, 200 mg/mL silica, Day28, bio rep 1


title: male 31 sperm, fresh

description: Library name: BB_31_Fresh1


title: Lungs, 200 mg/mL silica, Day14, bio rep 5

value: Day 14


title: Non cancer control 36 (Discovery cohort)

description: Library name: WS-18

value: Non cancer control


title: Lungs, 200 mg/mL silica, Day14, bio rep 4


title: male 27 sperm, fresh

description: Library name: BB_27_Fresh1


title: Lungs, 200 mg/mL silica, Day14, bio rep 2


title: Lungs, 200 mg/mL silica, Day14, bio rep 1


title: male 25 sperm, fresh

description: Library name: BB_25_Fresh1


title: Lungs, normal saline, Day28, bio rep 6

value: normal saline


title: Non cancer control 33 (Discovery cohort)

description: Library name: WS-15


title: Non cancer control 35 (Discovery cohort)

description: Library name: WS-17


title: Lungs, 200 mg/mL silica, Day14, bio rep 6


title: male 36 sperm, fresh

description: Library name: BB_36_Fresh1


title: Lungs, normal saline, Day28, bio rep 5


title: male 24 sperm, fresh

description: Library name: BB_24_Fresh1


title: Non cancer control 32 (Discovery cohort)

description: Library name: WS-14


title: Lungs, normal saline, Day28, bio rep 3


title: Lungs, normal saline, Day28, bio rep 2


title: male 23 sperm, fresh

description: Library name: BB_23_Fresh1


title: Lungs, normal saline, Day28, bio rep 1


title: male 22 sperm, fresh

description: Library name: BB_22_Fresh1


title: Cancer patient 2 (Validation cohort 1)

description: Library name: NL-17


title: Non cancer control 30 (Discovery cohort)

description: Library name: WS-12


title: Lungs, normal saline, Day14, bio rep 6


title: Lungs, normal saline, Day14, bio rep 5


title: male 20 sperm, fresh

description: Library name: BB_20_Fresh1


title: male 28 sperm, fresh

description: Library name: BB_28_Fresh1


title: Lungs, 200 mg/mL silica, Day14, bio rep 3


title: Lungs, normal saline, Day14, bio rep 4


title: Non cancer control 29 (Discovery cohort)

description: Library name: WS-11


title: Non cancer control 34 (Discovery cohort)

description: Library name: WS-16


title: Lungs, normal saline, Day14, bio rep 3


title: male 17 sperm, fresh

description: Library name: BB_17_Fresh1


title: Lungs, normal saline, Day14, bio rep 2


title: Non cancer control 28 (Discovery cohort)

description: Library name: WS-10


title: male 16 sperm, fresh

description: Library name: BB_16_Fresh1


title: Lungs, normal saline, Day28, bio rep 4


title: Lungs, normal saline, Day14, bio rep 1


title: Non cancer control 27 (Discovery cohort)

description: Library name: WS-09


title: male 15 sperm, fresh

description: Library name: BB_15_Fresh1


title: humanTumor21

city: Tampa

first: Antonio

last: Amelio

state: Fl

address: 12902 USF Magnolia Drive

department: Tumor Biology

institute: Moffitt Cancer Center

data_processing:
Paired-end reads were trimmed of adaptors and low-quality bases via Base-calling software fastp v0.23.2
Trimmed fastq files were aligned to the GRCh38.d1.vd1 version of the human genome with GENCODE v36 version of the human transcriptome using STAR 2.7.6a alignment software
Assembly: GRCh38.d1.vd1
Supplementary files format and content: Fastq raw reads files for each sample

source_name: parotid salivary gland

growth_protocol: Sample Isolation protocol: FFPE tissue samples were sent to the UNC Lineberger Comprehensive Cancer Center Translational Genomics Lab (TGL) for RNA isolation using the Maxwell 16 MDx Instrument (Promega AS3000) and the Maxwell 16 LEV RNA FFPE Kit (Promega AS1260) according to the manufacturer’s protocol (Promega 9FB167).

extract_protocol:
For human salivary MEC samples, total RNA sequencing libraries were prepared at TGL using a Bravo Automated Liquid-Handling Platform (Agilent G5562A) and the TruSeq Stranded Total RNA Library Prep Gold Kit (Illumina 20020599) according to the manufacturer’s protocol (Illumina 1000000040499).
RNAseq library quality and quantity were measured using a TapeStation 4200 (Agilent G2991AA), pooled at equimolar ratios and denatured according to the manufacturer’s protocol (Illumina 15050107). Sequencing was performed at the High Throughput Sequencing Facility (HTSF) at UNC Chapel Hill.
Two RNAseq libraries were sequenced per lane on a HiSeq2500 (Illumina SY–401–2501) with 2x50 bp paired-end configuration according to the manufacturer’s protocol (Illumina 15035786).

treatment_protocol: Pathology review of an H&E-stained slide was used to guide macro-dissection of unstained slides to enrich for tumor RNA.

value: parotid salivary gland

value: F

tag: tumor grade

value: High

tag: crtc1/maml2 fusion_status

value: Positive


title: Non cancer control 31 (Discovery cohort)

description: Library name: WS-13


title: humanTumor20

value: Negative


title: male 12 sperm, fresh

description: Library name: BB_12_Fresh1

value: sperm

value: fresh


title: Non cancer control 26 (Discovery cohort)

description: Library name: WS-08


title: Non cancer control 25 (Discovery cohort)

description: Library name: WS-07


title: humanTumor18

value: M


title: humanTumor19

value: not provided


title: humanTumor17


title: male 11 sperm, fresh

description: Library name: BB_11_Fresh1


title: humanTumor16


title: humanTumor15


title: male 9 sperm, fresh

description: Library name: BB_9_Fresh1


title: humanTumor14


title: DriverMap AIR TCR RNA CDR3 UMI-based (human, high input)

city: Mountain View

first: Kitt

middle: D

last: Paraiso

address: 320 Logue Ave.

department: Bioinformatics

institute: Cellecta, Inc.

description:
Library name: R_T-CDR3-UMI-50ng
Template input: 50 ng

data_processing:
Sample Demultiplexing: bcl2fastq was used to demultiplex and convert the sequencing results to individual sample fastqs.
QC Analysis: fastqc was used to assess the quality of the sequencing reads prior to downstream analysis.
Read Alignment: MiXCR was used to align the reads to the reference immune repertoire genes. This command also geneartes the clonotype tables along with relevant metadata information like read counts, aligning V(D)J genes, etc. The command used: mixcr analyze local:cellecta-new --species $SPECIES --assemble-clonotypes-by $ASSEMBLY_TYPE $SPLIT_CLONES_CONFIG --$MOLECULE --tag-pattern $TAG_PATTERN --rigid-left-alignment-boundary --floating-right-alignment-boundary $BOUNDARY --export-productive-clones-only $READ_1 $READ_2 $OUTPUT_NAME; where $SPECIES = hsa (human) or mmu (mouse), $ASSEMBLY_TYPE = CDR3 (CDR3 amplicon) or {CDR1Begin:FR4End} (Full length amplicon), $SPLIT_CLONES_CONFIG = --split-clones-by C (if working with B cells to distinguish isotypes) or otherwise skip this option, $MOLECULE = rna (for RNA) or dna (for DNA), $BOUNDARY = J (CDR3 amplicon) or C (Full length amplicon), $READ_1, $READ_2 = paired end read file names (e.g. sample_a_R1.fastq.gz, sample_a_R2.fastq.gz), $OUTPUT_NAME = name of output files (e.g. sample_a) and $TAG_PATTERN = '^(R1:*)\^(MIVBC:GCATCAN)(R2:*)|^(MIVBC:AGTCGTN)(R2:*)|^(MIVBC:TCGCATCN)(R2:*)|^(MIVBC:AGCGTAGN)(R2:*)|^(MIVBC:TACGACTN)(R2:*)|^(MIVBC:CTGATGAN)(R2:*)|^(MIVBC:CATAGCATN)(R2:*)|^(MIVBC:GTAGGCTAN)(R2:*)'. The preset is specified by a cellecta-new.yaml file.
Template Estimation: The number of molecule templates is estimated using a normalization factor. Briefly, rare clonotypes are identified from each dataset. The average number of reads among the rare clonotypes is used as the normalization factor. This value is used as the quotient to convert read counts to the estimated number of molecule templates.
Further clarification on the data processing can be found on the DriverMap AIR protocol (https://cellecta.com/pages/product-user-manuals).
Assembly: The sequences were aligned to MiXCR's custom curated set of V, D, J and C genes in MiXCR version v4.7.0.
Supplementary files format and content: The clones tables contain information on the clonotypes in the repertoire including read counts, gene usage, and estimated number of templates.
Library strategy: TCR-Seq

source_name: PBMC

extract_protocol:
RNA extraction: QIAGEN RNAeasy Micro/Mini Plus Kit for total RNA isolation. DNA extraction: DNeasy Blood & Tissue kit was used for DNA extraction.
AIR-RNA Protocol 1. Hybridization of RevGSP with RNA. 50ng or 10ng of RNA were hybridized with mix of Reverse TCR or BCR (C-region) gene-specific primers (GSPs) in 20ul of 1xHyb buffer at 70C, 5 min, 60C for 60 min, cool down to 25C, add 24 ul (1.2V) of SPRI beads, put the test tube in magnetic stand and wash the beads twice with 200ul of 80% ethanol. 2. Rev GSP extension > cDNA synthesis. The washed magnetic beads were resuspended in 22 ul of 1xRT-extension buffer, remove beads in magnetic stand, collect 20.5 ul of supernatant and incubated at 50C for 35 min, and 72C for 10min. 3. Fwd GSP extension. cDNA product (in 20 ul) was extended by adding 20 ul of DNA polymerase master mix with pool of mouse Forward TCR or BCR FR1 or FR3 GSPs, incubated 98C, 1min, 68C, 10 min and treated with 0.5 ul of primer removal enzyme mix (10x) at 37C, 30min, 95C,5min.  4. 1st PCR. FwdGSP-extended cDNA was diluted in PCR master mix (60ul) with universal anchor primers, and anchored cDNA fragments were amplified in 100-ul of Multiplex DNA polymerase reaction mix with universal anchor PCR primers for 17 cycles (50ng RNA), or 20 cycles (10ng RNA).  5. 2nd PCR.  2ul aliquots of 1st PCR were added in 50ul master mix for 2nd PCR step, amplified using unique combination of Illumina’s UDP Fwd-P5-Index and UDP Rev-P7-Index primers for 8 cycles, add extra 1 cycle for AIR-TCR and 3 cycles for AIR-mTCR and AIR-mBCR and treated with primer removal enzyme mix (1-ul) at 37C for 30min. PCR products were analyzed in 3% agarose gel and Fragment Analyzer, combined at 1.5x for FR1 products and ½ for 10ng products amount, purified using AMPpure magnetic beads (1.5X volume). The purified cDNA products were quantitated by Qubit fluorescence measurement, and diluted to 10 nM (1.6 ng/ul) for next-generation sequencing using NextSeq500.    AIR-DNA Protocol 1. RevGSP extension. DNA samples(2ug or 0.4ug) were extended with pool of Reverse AIR TCR or BCR J region GSPs in 50 ul of PCR master mix, incubated at 98C, 1min, 68C, 10 min and treated with 0.5 ul of primer removal enzyme mix (10x), at 37C, 20min, 95C,5min.  2. Fwd GSP extension. Rev GSP-extended DNA product (in 50 ul) was extended by adding 2.5ul of pool of Forward AIR TCR or BCR FR3 GSPs, incubated at 98C, 1min, 68C, 10 min and treated with 0.5 ul of primer removal enzyme mix (10x), at 37C, 20min, 95C,5min.  3. 1st PCR. FwdGSP-extended DNA was diluted in PCR master mix (50ul), and anchored cDNA fragments were amplified in 100-ul of Multiplex DNA polymerase reaction mix with universal anchor PCR primers for 22 (TCR-2ug), 25 (TCR-0.4ug), 25 (BCR-2ug) or 28 (BCR-0.4ug)cycles: (98C, 20sec, 65C, 10sec, 72C,30sec).  4. 2nd PCR.  2ul aliquotes of 1st PCR were added in 50ul of master mix for 2nd PCR step (each tube has unique Dual Index primers) and amplified using unique combination of Fwd-P5-Index and Rev-P7-Index for 8 cycles, treated with primer removal enzyme mix (1-ul) at 37C for 30min. PCR products were analyzed in Agarose gel and Fragment analyzer, combined together at 2x (2ug) and 1x (0.4ug), mix with AIR RNA samples and purified by AMPure beads. The purified cDNA products were quantitated by Qubit fluorescence measurement, and diluted to 10 nM (1.6 ng/ul) for next-generation sequencing using NextSeq500.    Further clarification on the library construction can be found on the DriverMap AIR protocol (https://cellecta.com/pages/product-user-manuals).

value: PBMC

value: Healthy Donor

value: T Cell

value: WT

tag: batch


title: Non cancer control 23 (Discovery cohort)

description: Library name: WQ-05

value: plasma


title: male 8 sperm, fresh

description: Library name: BB_8_Fresh1


title: Non cancer control 22 (Discovery cohort)

description: Library name: EO-06


title: DriverMap AIR BCR RNA CDR3 UMI-based (human, high input)

description:
Library name: R_B-CDR3-UMI-50ng
Template input: 50 ng

data_processing:
Sample Demultiplexing: bcl2fastq was used to demultiplex and convert the sequencing results to individual sample fastqs.
QC Analysis: fastqc was used to assess the quality of the sequencing reads prior to downstream analysis.
Read Alignment: MiXCR was used to align the reads to the reference immune repertoire genes. This command also geneartes the clonotype tables along with relevant metadata information like read counts, aligning V(D)J genes, etc. The command used: mixcr analyze local:cellecta-new --species $SPECIES --assemble-clonotypes-by $ASSEMBLY_TYPE $SPLIT_CLONES_CONFIG --$MOLECULE --tag-pattern $TAG_PATTERN --rigid-left-alignment-boundary --floating-right-alignment-boundary $BOUNDARY --export-productive-clones-only $READ_1 $READ_2 $OUTPUT_NAME; where $SPECIES = hsa (human) or mmu (mouse), $ASSEMBLY_TYPE = CDR3 (CDR3 amplicon) or {CDR1Begin:FR4End} (Full length amplicon), $SPLIT_CLONES_CONFIG = --split-clones-by C (if working with B cells to distinguish isotypes) or otherwise skip this option, $MOLECULE = rna (for RNA) or dna (for DNA), $BOUNDARY = J (CDR3 amplicon) or C (Full length amplicon), $READ_1, $READ_2 = paired end read file names (e.g. sample_a_R1.fastq.gz, sample_a_R2.fastq.gz), $OUTPUT_NAME = name of output files (e.g. sample_a) and $TAG_PATTERN = '^(R1:*)\^(MIVBC:GCATCAN)(R2:*)|^(MIVBC:AGTCGTN)(R2:*)|^(MIVBC:TCGCATCN)(R2:*)|^(MIVBC:AGCGTAGN)(R2:*)|^(MIVBC:TACGACTN)(R2:*)|^(MIVBC:CTGATGAN)(R2:*)|^(MIVBC:CATAGCATN)(R2:*)|^(MIVBC:GTAGGCTAN)(R2:*)'. The preset is specified by a cellecta-new.yaml file.
Template Estimation: The number of molecule templates is estimated using a normalization factor. Briefly, rare clonotypes are identified from each dataset. The average number of reads among the rare clonotypes is used as the normalization factor. This value is used as the quotient to convert read counts to the estimated number of molecule templates.
Further clarification on the data processing can be found on the DriverMap AIR protocol (https://cellecta.com/pages/product-user-manuals).
Assembly: The sequences were aligned to MiXCR's custom curated set of V, D, J and C genes in MiXCR version v4.7.0.
Supplementary files format and content: The clones tables contain information on the clonotypes in the repertoire including read counts, gene usage, and estimated number of templates.

value: B Cell


title: humanTumor13


title: male 6 sperm, fresh

description: Library name: BB_6_Fresh1


title: humanTumor22


title: Non cancer control 21 (Discovery cohort)

description: Library name: EO-05

value: Non cancer control


title: DriverMap AIR TCR DNA CDR3 UMI-based (human, high input)

description:
Library name: D_T-CDR3-UMI-2ug
Template input: 2 ug

library_source: genomic

molecule: genomic DNA


title: male 5 sperm, fresh

description: Library name: BB_5_Fresh1


title: humanTumor11


title: Non cancer control 19 (Discovery cohort)

description: Library name: EO-03


title: DriverMap AIR BCR DNA CDR3 UMI-based (human, high input)

description:
Library name: D_B-CDR3-UMI-2ug
Template input: 2 ug


title: male 4 sperm, fresh

description: Library name: BB_4_Fresh1


title: Non cancer control 20 (Discovery cohort)

description: Library name: EO-04


title: DriverMap AIR TCR RNA Full Length VBC-based (human, high input)

description:
Library name: R_T-CDR123-VBC-50ng
Template input: 50 ng


title: humanTumor10


title: Non cancer control 24 (Discovery cohort)

description: Library name: WQ-11


title: male 3 sperm, fresh

description: Library name: BB_3_Fresh1


title: Non cancer control 18 (Discovery cohort)

description: Library name: EO-02


title: Non cancer control 17 (Discovery cohort)

description: Library name: EM-12


title: male 1 sperm, fresh

description: Library name: BB_1_Fresh1


title: DriverMap AIR TCR RNA Full Length VBC-based (human, low input)

description:
Library name: R_T-CDR123-VBC-10ng
Template input: 10 ng


title: humanTumor9


title: DriverMap AIR BCR RNA Full Length VBC-based (human, high input)

description:
Library name: R_B-CDR123-VBC-50ng
Template input: 50 ng


title: DriverMap AIR BCR RNA Full Length VBC-based (human, low input)

description:
Library name: R_B-CDR123-VBC-10ng
Template input: 10 ng


title: humanTumor7

value: Low


title: Non cancer control 16 (Discovery cohort)

description: Library name: EM-11


title: male 44 sperm, cryopreserved

description: Library name: BB_44_CryoP1

value: cryopreserved


title: DriverMap AIR TCR RNA CDR3 VBC-based (human, high input)

description:
Library name: R_T-CDR3-VBC-50ng
Template input: 50 ng


title: Non cancer control 15 (Discovery cohort)

description: Library name: EM-09


title: humanTumor12

value: parotid salivary gland


title: DriverMap AIR TCR RNA CDR3 VBC-based (human, low input)

description:
Library name: R_T-CDR3-VBC-10ng
Template input: 10 ng


title: Non cancer control 14 (Discovery cohort)

description: Library name: EM-07


title: male 42 sperm, cryopreserved

description: Library name: BB_42_CryoP1


title: humanTumor5

value: ND


title: DriverMap AIR BCR RNA CDR3 VBC-based (human, high input)

description:
Library name: R_B-CDR3-VBC-50ng
Template input: 50 ng


title: male 41 sperm, cryopreserved

description: Library name: BB_41_CryoP1


title: male 2 sperm, fresh

description: Library name: BB_2_Fresh1


title: Non cancer control 12 (Discovery cohort)

description: Library name: EM-05


title: humanTumor6

value: F

value: Positive


title: humanTumor8


title: humanTumor3


title: male 39 sperm, cryopreserved

description: Library name: BB_39_CryoP1

value: sperm


title: Non cancer control 11 (Discovery cohort)

description: Library name: EM-04


title: DriverMap AIR BCR RNA CDR3 VBC-based (human, low input)

description:
Library name: R_B-CDR3-VBC-10ng
Template input: 10 ng


title: Non cancer control 13 (Discovery cohort)

description: Library name: EM-06


title: Non cancer control 10 (Discovery cohort)

description: Library name: EM-03


title: humanTumor1

value: Negative


title: DriverMap AIR TCR DNA CDR3 VBC-based (human, high input)

description:
Library name: D_T-CDR3-VBC-2ug
Template input: 2 ug


title: humanTumor2

value: M


title: DriverMap AIR TCR DNA CDR3 VBC-based (human, low input)

description:
Library name: D_T-CDR3-VBC-0p4ug
Template input: 0.4 pg


title: male 36 sperm, cryopreserved

description: Library name: BB_36_CryoP1


title: male 43 sperm, cryopreserved

description: Library name: BB_43_CryoP1


title: Non cancer control 9 (Discovery cohort)

description: Library name: EM-02


title: DriverMap AIR BCR DNA CDR3 VBC-based (human, high input)

description:
Library name: D_B-CDR3-VBC-2ug
Template input: 2 ug


title: humanNormal6

value: Normal


title: male 35 sperm, cryopreserved

description: Library name: BB_35_CryoP1


title: Non cancer control 8 (Discovery cohort)

description: Library name: EK-06


title: humanNormal5


title: male 34 sperm, cryopreserved

description: Library name: BB_34_CryoP1


title: humanTumor4


title: humanNormal4


title: Non cancer control 7 (Discovery cohort)

description: Library name: EK-05


title: DriverMap AIR TCR RNA CDR3 VBC-based (mouse, low input)

description:
Library name: R_mT-CDR3-VBC-10ng
Template input: 10 ng

value: PBMC


title: Non cancer control 6 (Discovery cohort)

description: Library name: EK-04


title: male 37 sperm, cryopreserved

description: Library name: BB_37_CryoP1


title: DriverMap AIR BCR RNA CDR3 VBC-based (mouse, low input)

description:
Library name: R_mB-CDR3-VBC-10ng
Template input: 10 ng

value: Healthy Donor


title: Non cancer control 5 (Discovery cohort)

description: Library name: EK-03


title: Single-cell suspension from collecting lymphatic vessels from female mice (F1-F4)_CMO

city: New Orleans

first: Jorge

last: Castorena-Gonzalez

state: LA

address: 1430 Tulane Avenue, MC-8683

department: Pharmacology

institute: Tulane University

description:
Library name: Sample4_CMO
Age: 12-14 weeks of age.

data_processing:
Cell Ranger version 7.1.0 (10x Genomics Inc, CA) was used to map raw data to references of mouse genome mm10-2020-A. Analyses were performed using Seurat (4.3.0.1) in R studio (RStudio 2023.06.0+421). During quality control, cells with feature counts in the range 1000-6250 and cells displaying less than 5% mitochondrial counts were selected. Following quality control, the dataset underwent pipeline processing that included normalization using VST, multi-dimensional reduction and cell clustering, differential testing framework, and visualization.
Assembly: Mouse Genome mm10-2020-A
Supplementary files format and content: Processed files including: barcodes.tsv.gz, features.tsv.gz, and matrix.mtx.gz include the aggregated data from all female and male samples. Samples can be identified by their barcode trail indexes, i.e., -1 to -4 for female samples (F1, F2, F3, and F4) and -5 to -8 for male samples (M1, M2, M3, M4).

source_name: Collecting lymphatic vessels and surrounding tissues

extract_protocol:
Lymphatic vessels were carefully dissected as described in the previous section and placed in ice-cold low-calcium physiological saline solution (PSS) containing 1 mg/mL trypsin inhibitor, 0.1 mg/mL DNase I, 1 mM DTT, and 0.02 U/µL RNase inhibitor. For each mouse, lymphatic vessels from four anatomical regions (i.e., popliteal afferent (saphenous), inguinal axillary, mesenteric, and superficial cervical) were combined into a single sample. A two-step tissue dissociation process was utilized. Tissues were transferred into enzymatic solution part 1 containing 1 mg/mL trypsin inhibitor, 0.1 mg/mL DNase I, 1 mM DTT, 0.02 U/µL RNase inhibitor, and 1 mg/mL papain in low-calcium PSS, and incubated at 37°C for 30 min with intermittent shaking every 9 minutes for 1 minute at 800 rpm. Meanwhile, enzyme solution part 2 was prepared: 1 mg/mL trypsin inhibitor, 0.1 mg/mL DNase I, 1 mM DTT, 0.02 U/µL RNase inhibitor, 1 mg/mL papain, 2 mg/mL collagenase A, and 3 mg/mL dispase II in low-calcium PSS. After the 30-minute incubation in enzyme solution part 1, vessels were transferred into enzyme solution part 2 and incubated for an additional 30 min at 37°C with intermittent shaking every 7 minutes for 1 minute at 800 rpm. Single-cell suspensions were ensured via pipetting up and down with a P200 pipette and a fine-tip Pasteur glass pipette. The volume of the single cell suspensions was doubled with cold low-calcium PSS to lower concentration of enzymes and stop/minimize enzymatic activity, and then cell suspensions were centrifuged at 600xg for 8 min at 4°C. Following centrifugation, supernatants were carefully removed, and cell pellets were gently resuspended with 200 µL sterile PBS containing 0.04% ultra-pure BSA. Cells were strained into a 1.5 mL LoBind centrifuge tube and cell counts and viability were assessed.
Feature Barcode Technology and Single Cell RNA Sequencing. Cell numbers and viability were validated by Cellometer Automated Cell Counter (Nexcelom Bioscience, Lawrence, MA, USA) prior to scRNAseq library preparation. For feature barcode technology and 10x scRNAseq assay, individual live cells per sample were targeted using feature barcode oligonucleotide conjugated to a lipid. Labeled cells from each sample were pooled and 10x Single Cell 3’ RNAseq technology was applied to generate both cell multiplexing libraries and cDNA libraries. Briefly, single cells from each sample were labeled with unique cell multiplexing oligo (CMO) provided by 10x Genomics (10X Genomics Inc, CA). 5,000 labeled cells from each sample were pooled and partitioned into nanoliter-scale Gel Beads-In-EMulsion (GEMs). Feature barcode molecules were captured directly by oligonucleotides presented on the Gel Beads inside a GEM during GEM-RT. Cell multiplexing libraries comprising dual index NN set A (10X Genomics Inc, CA) were generated by index PCR amplification. Meanwhile, full-length barcoded cDNAs were also generated and amplified by PCR to obtain sufficient mass for library construction. Following enzymatic fragmentation, end-repair, A-tailing, and adaptor ligation, single cell 3’ libraries comprising dual index TT set A (10X Genomics Inc, CA) were generated. Library quality controls were performed by using Agilent High Sensitive DNA kit with Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA) and quantified by Qubit 2.0 fluorometer (Thermo Fisher Scientific). Pooled libraries at a final concentration of 750pM were sequenced with paired end dual index configuration by Illumina NextSeq 2000.


title: male 31 sperm, cryopreserved

description: Library name: BB_31_CryoP1


title: Single-cell suspension from collecting lymphatic vessels from male mice (M1-M4)_CMO

description:
Library name: Sample3_CMO
Age: 12-14 weeks of age.

value: Collecting lymphatic vessels and surrounding tissues


title: male 28 sperm, cryopreserved

description: Library name: BB_28_CryoP1


title: humanNormal3


title: humanNormal2


title: humanNormal1


title: male 25 sperm, cryopreserved

description: Library name: BB_25_CryoP1


title: Non cancer control 3 (Discovery cohort)

description: Library name: EK-01


title: Single-cell suspension from collecting lymphatic vessels from female mice (F1-F4)_GEX

description:
Library name: Sample2_GEX
Age: 12-14 weeks of age.

library_source: transcriptomic single cell


title: male 24 sperm, cryopreserved

description: Library name: BB_24_CryoP1


title: Non cancer control 2 (Discovery cohort)

description: Library name: EI-04


title: Single-cell suspension from collecting lymphatic vessels from male mice (M1-M4)_GEX

description:
Library name: Sample1_GEX
Age: 12-14 weeks of age.


title: male 23 sperm, cryopreserved

description: Library name: BB_23_CryoP1


title: DriverMap AIR BCR DNA CDR3 VBC-based (human, low input)

description:
Library name: D_B-CDR3-VBC-0p4ug
Template input: 0.4 pg

value: WT


title: Non cancer control 1 (Discovery cohort)

description: Library name: EI-01

value: plasma


title: clone Sox2P-reporter mC-mT-cl9 Rv

city: Amsterdam

first: Christina

last: Moene

address: Plesmanlaan 121

country: Netherlands

institute: Netherlands Cancer Institute

description:
Library name: E2709 clone Sox2P-reporter mC-mT-cl9 Rv
clone Sox2P-reporter mC-mT-cl9
mapped_integrations_Sox2P_clones.txt

data_processing:
The full protocol is described in the corresponding manuscript. In short, reads overlapping the SB-genome junction were selected, SB-sequences were trimmed off (using an adapted version of cutadapt) and reads were mapped to the genome (mm10 or an in silico version of mm10 including the hopping launch pad at -116 kb), filtering for mapq >=10. All putative integrations are lifted over to mm10.
Assembly: mm10
Supplementary files format and content: The processed data file reports all putative integrations mapped in a pool or clone (supported by at least one mapping read, filtering in the manuscript can be more strict). Columns: chr = chromosome; start, end = integration location; seq = dinucleotide at integration site (expected for SB: TA); strand = strand of integration (orientation); read_count = total read count, mapq = total mapping quality (of all reads); read_count_1/mapq_1 = forward mapping reads (3' ITR); read_count_2/mapq_2 = reverse mapping reads (5' ITR); all other columns specify and annotate the sequenced sample. There is one processed data file per founder cell line (pool-based experiments) and one for the isolated clones.

source_name: F1 hybrid 129/Sv:CAST/EiJ

growth_protocol: F121/9 (CAST/EiJ x S129/Sv) Sox2 mCherry+/eGFP+ were cultured in Serum+Lif+2i condition. Briefly, cells were cultured on gelatin-coated (0.1%) culture plates in Glasgow minimum essential medium (Sigma-Aldrich, G5154) supplemented with 15% fetal bovine serum (Thermo Fisher Scientific, 10270-106), 1% L-glutamine (Thermo Fisher Scientific, 25030024), 1% sodium pyruvate (Thermo Fisher Scientific, 11360-70), 1% MEM non-essential amino acids (Thermo Fisher Scientific, 11140-50), 1% penicillin-streptomycin (Thermo Fisher Scientific, 15070063), 100 µM ß-mercaptoethanol, 10 x 4 U leukemia inhibitory factor (LIF; esg1107, Millipore), 1 μM MEK inhibitor PD0325901 (Mirdametinib, MedChemExpress), 3 μM GSK-3β inhibitor CHIR99021 (Laduviglusib, MedChemExpress) in 5% CO2 at 37°C. Cells were passaged every 2 days.

extract_protocol:
Sorted pools and clones were expanded and crude lysates were obtained from a full 96 well plate by lysing with 50 µL DirectPCR Lysis Reagent (102-T, Viagen Biotec) supplemented with 100 µg/mL proteinase K, and incubating 2.5 h at 55 °C and 45 min at 85 °C. For unsorted control cell pools, gDNA was extracted after expansion using the ISOLATE II Genomic DNA Kit (BIO-52067, Bioline).
SBt integrations were mapped by amplifying and sequencing the ITR-genome junction using a tagmentation-based approach. The procedure was similar to {Schep, 2021, 33848455}, but using the following SB-ITR specific primers: MEP009 (5′ ITR, reverse) or LD027 (3′ ITR, forward) for the linear enrichment and MEP011 (5′ ITR, reverse) or MEP034 (3′ ITR, forward) for PCR.Input for one tagmentation reaction was either 100ng gDNA or 3 µL crude lysate. The 72 °C extension times of all PCRs were increased to 1 min. In addition, the composition of the linear enrichment PCR was changed to 10 μL of tagmented DNA, 2 μL of 1 μM primer, 4 μL dNTPs (10mM), 8 μL 5x Phusion® HF Buffer (NEB), 0.5 μL Phusion® HS Flex polymerase (2 U/μL - NEB), in a final volume of 40 μL. For all clones and some experiments with sorted pools (proof-of-principle experiment, third replicate of the reporter hopping) we halved this reaction to a final volume of 20 µL. For the unsorted control pools, 10 separate tagmentation reactions and library preparations were performed, usually using one set of sequencing indices. Libraries were sequenced with 150 bp paired-end reads using either an Illumina MiSeq system (reagent kit v2) or  Illumina NextSeq 550 system (mid-output kit) including 10% of PhiX spike-in.  Primers are described in the corresponding manuscript.

treatment_protocol: SB hopping was induced by transfecting 2.5 to 5 million cells with the bicistronic plasmid (pME07) encoding SB transposase SB100x and the human nerve growth factor receptor (LNGFR), using Lipofectamine 2000 (Invitrogen, 11668019) and 5 µg of plasmid per million cells. After 30 hours, transfected cells were enriched using MACS MS columns (130-042-201, Miltenyi Biotec) and LNGFR MicroBeads (130-091-330, Miltenyi Biotec) according to the manufacturer’s protocol. One week after transfection, 0.2 million SB-transposase-transfected cells were set apart as an unsorted pool and the remaining cells were FACS sorted into pools (of up to 1000 cells) based on reporter expression. Clones were obtained by sorting single cells from previously obtained pools, or sorted directly from the full pool of SB-transposase-transfected cells.

value: F1 hybrid 129/Sv:CAST/EiJ

value: mESC

tag: launch pad_location

tag: insert

tag: sorted gate

tag: sample type

value: deletion clone


title: LP-161kb_Sox2P_CDS_mChDel_CM2291_rep2

description: Library name: LP-161kb_Sox2P_CDS_mChDel_CM2291_rep2

data_processing:
RCMC data analysis was performed in an allele-specific manner using custom 129S1/CAST F1-hybrid genomes. To construct the 129S1/CAST genome, mouse strain-specific variants were obtained from the Mouse Genomes Project (PMID: 21921910), homozygous SNPs were filtered using SnpSift v4.3p (PMID: 22728672), and bcftools v1.9 (PMID: 33590861) was used to insert single nucleotide variants into the GRCm38/mm10 mouse genome obtained from the Ensembl database. The resulting hybrid genome was further modified to generate custom genomes for each of the cell lines used in the RCMC (F121_Sox2_GFP/mCherry; LP-116 kb_Sox2P; LP-161 kb_Sox2P; LP-116 kb Sox2P_mChDel; LP-161 kb_Sox2P_mChDel; LP-161 kb_Sox2P_CDS_mChDel). To do this, the Sox2P or Sox2P-CDS inserts were added at -116 kb or -161 kb launch pad coordinates on the 129S1 allele. In order to unify the size of the chromosomes, the corresponding locus of the CAST allele was filled with N-stretches. If the editing was absent in the cell line or the Sox2 gene deletion was present, the corresponding genomic regions of 129S1 allele were additionally modified with N-stretches. In addition, the mCherry and GFP tag were added to the Sox2 gene on the 129S1 and CAST allele respectively. RCMC paired-end reads were aligned to the custom hybrid 129S1/CAST genomes using bwa-mem v0.7.17-r1188 (PMID: 19451168) with the "-SP" options. The aligned reads were processed using pairtools v1.1.2 (PMID: 36824968) following the haplotype-resolved guideline from the pairtools. First, the reads were parsed into contact pairs using pairtools parse with the parameters "--add-columns mapq,XA,NM,AS,XS", "--walks-policy mask" and "--min-mapq 0". Secondly, the pairs were phased using pairtools phase in "--tag-mode XA". Finally, pairtools dedup was used with the parameters "--backend cython" and "--max-mismatch 1" to remove duplicates. The remaining reads were filtered to contain only pairs where at least one end was phased and both ends originated from the captured locus of interest, to avoid downstream biases described in the original RCMC study (PMID: 37157000). These resulting reads were then converted to MCOOL format and iteratively corrected using cooler v0.10.3 (PMID: 31290943)
Assembly: mm10 (custom genome build)
Supplementary files format and content: mcool files containing contact pairs of at least one phased end read and both reads coming from the capture locus. Allele specific mcool files per replicate and for combined replicates are provided.

extract_protocol:
Region-capture Micro-C (RCMC) was performed as previously described (Goel et al. 2023 PMID: 37157000). Briefly, nuclei were extracted from crosslinked cells and the chromatin digested with micrococcal nuclease into nucleosome-sized fragments. These fragments were labeled with biotinylated nucleotides and ligated by T4 DNA ligase to other spatially proximal fragments overnight. Crosslinks were reversed and dinucleosome-sized fragments were selected by electrophoresis on a 1% agarose gel and purified by pulldown on T1 streptavidin beads (Invitrogen).
Purified dinucleosome-sized fragments were prepared for Illumina sequencing using an NEBNext Ultra II library prep kit (NEB #E7645S) following manufacturer’s instructions. Prepared libraries on beads were amplified by PCR using Q5 DNA polymerase to generate sufficient input for target enrichment. Prior to sequencing, the target region was enriched via binding to biotinylated DNA probes and streptavidin bead pulldown (Twist). The enriched library was PCR amplified using Equinox library amplification mix; the minimum number of cycles to generate sufficient input for sequencing was performed. Libraries were sequenced on an Illumina NovaSeq X.

treatment_protocol: 25x10^6 cells were fixed in 3mM disuccinimidyl glutarate for 35 minutes; 1% formaldehyde was added and crosslinking continued for 10 minutes. Fixed cells were split into aliquots of 5 million cells.

value: Sox2::mCherry deleted


title: LP-161kb_Sox2P_CDS_mChDel_CM2291_rep1

description: Library name: LP-161kb_Sox2P_CDS_mChDel_CM2291_rep1


title: LP-161kb_Sox2P_mChDel_CM2266_rep2

description: Library name: LP-161kb_Sox2P_mChDel_CM2266_rep2


title: male 27 sperm, cryopreserved

description: Library name: BB_27_CryoP1

value: cryopreserved


title: clone Sox2P-reporter mC-mT-cl7 Rv

description:
Library name: E2709 clone Sox2P-reporter mC-mT-cl7 Rv
clone Sox2P-reporter mC-mT-cl7
mapped_integrations_Sox2P_clones.txt


title: male 22 sperm, cryopreserved

description: Library name: BB_22_CryoP1


title: Cancer patient 26 (Discovery cohort)

description: Library name: WS-25

value: Cancer patient


title: male 20 sperm, cryopreserved

description: Library name: BB_20_CryoP1


title: LP-161kb_Sox2P_mChDel_CM2266_rep1

description: Library name: LP-161kb_Sox2P_mChDel_CM2266_rep1


title: clone Sox2P-reporter mC-mT-cl2 Rv

description:
Library name: E2709 clone Sox2P-reporter mC-mT-cl2 Rv
clone Sox2P-reporter mC-mT-cl2
mapped_integrations_Sox2P_clones.txt


title: clone Sox2P-reporter mC-mT-cl5 Rv

description:
Library name: E2709 clone Sox2P-reporter mC-mT-cl5 Rv
clone Sox2P-reporter mC-mT-cl5
mapped_integrations_Sox2P_clones.txt


title: Non cancer control 4 (Discovery cohort)

description: Library name: EK-02

value: Non cancer control


title: male 17 sperm, cryopreserved

description: Library name: BB_17_CryoP1


title: LP-161kb_Sox2P_CM2094_rep2

description: Library name: LP-161kb_Sox2P_CM2094_rep2

value: WT, Sox2::GFP/mCh+


title: Cancer patient 25 (Discovery cohort)

description: Library name: WS-06


title: LP-116kb_Sox2P_mChdel_CM2267_rep2

description: Library name: LP-116kb_Sox2P_mChdel_CM2267_rep2


title: clone Sox2P-reporter mC-mTctrl2 Rv

description:
Library name: E2709 clone Sox2P-reporter mC-mTctrl2 Rv
clone Sox2P-reporter mC-mTctrl2
mapped_integrations_Sox2P_clones.txt


title: LP-161kb_Sox2P_CM2094_rep1

description: Library name: LP-161kb_Sox2P_CM2094_rep1


title: male 15 sperm, cryopreserved

description: Library name: BB_15_CryoP1

value: sperm


title: male 12 sperm, cryopreserved

description: Library name: BB_12_CryoP1


title: Cancer patient 23 (Discovery cohort)

description: Library name: WS-03


title: Cancer patient 22 (Discovery cohort)

description: Library name: WS-02


title: clone Sox2P-reporter mC-mTctrl1 Rv

description:
Library name: E2709 clone Sox2P-reporter mC-mTctrl1 Rv
clone Sox2P-reporter mC-mTctrl1
mapped_integrations_Sox2P_clones.txt


title: LP-116kb_Sox2P_mChdel_CM2267_rep1

description: Library name: LP-116kb_Sox2P_mChdel_CM2267_rep1


title: male 11 sperm, cryopreserved

description: Library name: BB_11_CryoP1


title: LP-116kb_Sox2P_CM2167_rep2

description: Library name: LP-116kb_Sox2P_CM2167_rep2


title: Cancer patient 21 (Discovery cohort)

description: Library name: WS-01


title: Cancer patient 27 (Discovery cohort)

description: Library name: WS-26


title: clone Sox2P-reporter mC-mT-cl7 Fw

description:
Library name: E2709 clone Sox2P-reporter mC-mT-cl7 Fw
clone Sox2P-reporter mC-mT-cl7
mapped_integrations_Sox2P_clones.txt


title: clone Sox2P-reporter mC-mT-cl9 Fw

description:
Library name: E2709 clone Sox2P-reporter mC-mT-cl9 Fw
clone Sox2P-reporter mC-mT-cl9
mapped_integrations_Sox2P_clones.txt


title: male 9 sperm, cryopreserved

description: Library name: BB_9_CryoP1


title: Cancer patient 20 (Discovery cohort)

description: Library name: WQ-14


title: clone Sox2P-reporter mC-mT-cl5 Fw

description:
Library name: E2709 clone Sox2P-reporter mC-mT-cl5 Fw
clone Sox2P-reporter mC-mT-cl5
mapped_integrations_Sox2P_clones.txt


title: clone Sox2P-reporter mC-mT-cl2 Fw

description:
Library name: E2709 clone Sox2P-reporter mC-mT-cl2 Fw
clone Sox2P-reporter mC-mT-cl2
mapped_integrations_Sox2P_clones.txt


title: WT_Sox2_GFP+mCh+_CM1846_rep2

description: Library name: WT_Sox2_GFP+mCh+_CM1846_rep2


title: Cancer patient 24 (Discovery cohort)

description: Library name: WS-04


title: male 8 sperm, cryopreserved

description: Library name: BB_8_CryoP1


title: male 16 sperm, cryopreserved

description: Library name: BB_16_CryoP1


title: WT_Sox2_GFP+mCh+_CM1846_rep1

description: Library name: WT_Sox2_GFP+mCh+_CM1846_rep1


title: male 6 sperm, cryopreserved

description: Library name: BB_6_CryoP1


title: male 5 sperm, cryopreserved

description: Library name: BB_5_CryoP1


title: Cancer patient 18 (Discovery cohort)

description: Library name: WQ-12


title: clone Sox2P-reporter mC-mTctrl1 Fw

description:
Library name: E2709 clone Sox2P-reporter mC-mTctrl1 Fw
clone Sox2P-reporter mC-mTctrl1
mapped_integrations_Sox2P_clones.txt


title: clone Sox2P-reporter mC-mTctrl2 Fw

description:
Library name: E2709 clone Sox2P-reporter mC-mTctrl2 Fw
clone Sox2P-reporter mC-mTctrl2
mapped_integrations_Sox2P_clones.txt


title: Cancer patient 17 (Discovery cohort)

description: Library name: WQ-09


title: AAV_d14_3

city: Ann Arbor

first: Susan

middle: V

last: Brooks

state: Michigan

address: 109 Zina Pitcher Pl

department: Molecular and Integrative Physiology

institute: University of Michigan

description: Library name: Sample 23

data_processing:
For non-multiplexed samples, gene expression count matrices were generated using the count command from Cell Ranger v8.0.0 with the --include-introns=true argument, aligning reads to the GRCm39 mouse reference genome. For multiplexed samples labeled with CellPlex Cell Multiplexing Oligos (CMOs), count matrices were generated using the multi command in Cell Ranger v8.0.0, following the 10x Genomics guidelines for CMO demultiplexing. Default parameters were used unless otherwise specified.
Assembly: GRCm39
Supplementary files format and content: barcodes, features, and mtx files

molecule: nuclear RNA

source_name: Skeletal Muscle

extract_protocol:
Tibialis anterior muscles were harvested, minced, and nuclei were isolated using two approaches: (1) Dounce homogenization in Miltenyi Nuclei Extraction Buffer with RNase inhibitor, followed by filtration and centrifugation; or (2) the 10X Genomics Chromium Nuclei Isolation Kit, following the manufacturer’s protocol for frozen tissues. In both methods, nuclei were resuspended in wash buffer containing BSA and RNase inhibitor. Nuclei quality and counts were assessed by trypan blue exclusion with a hemocytometer or automated fluorescence-based cell counting. All steps were performed on ice or at 4°C to preserve RNA integrity.
Single-nucleus RNA-seq libraries were generated using the 10x Genomics Chromium Single Cell 3' Gene Expression v3.1 chemistry. Nuclei were loaded onto a Chromium Controller to create gel bead-in-emulsions (GEMs) for barcoding and reverse transcription. Libraries were constructed following the manufacturer’s protocol, either using the Standard or High-Throughput workflows. Some samples were multiplexed using 10x Genomics CellPlex Cell Multiplexing Oligos (CMOs) according to the CellPlex Protocol #1. Final libraries were sequenced on an Illumina NovaSeq X platform using a 2 × 150 bp configuration, targeting a depth of 50,000 reads per nucleus.

value: Skeletal Muscle

value: Skeletal muscle cells

value: AAV + 14 days post sciatic nerve crush

tag: library type

value: mRNA


title: Cancer patient 16 (Discovery cohort)

description: Library name: WQ-07


title: male 3 sperm, cryopreserved

description: Library name: BB_3_CryoP1


title: 4_3: Input Injury replicate 3

city: Los Angeles

first: Riki

last: Kawaguchi

address: 760 Westwood Plaza, Room 37-420

department: Department of Psychiatry and Neurology Department of Psychiatry

institute: University of California, Los Angeles

description: Input Injury replicate 3

data_processing:
Basecalls/demultiplexing performed using Illumina Bcl2fastq2 v 2.17
Alignment to mouse genome using STAR spliced read aligner
Counting reads using HTSeq with GRCm38 as reference
Normalization using trimmed mean of M values method, and differential expression analysis using Biocoductor packages.
Assembly: mm10
Supplementary files format and content: Counts

source_name: Dissociated L4-L6 DRG

extract_protocol:
RNA was extracted and purified using RNeasy Mini kit (Qiagen)
RNA-seq libraries were prepared with ribosomal-RNA-depletion based methods (Truseq Stranded Total RNA Library Prep Gold (Illumina)

value: Dissociated L4-L6 DRG

tag: condition

value: Input Injury


title: AAV_d0_3

description: Library name: Sample 21

tag: tissue

value: AAV uninjured


title: 4_2: Input Injury replicate 2

description: Input Injury replicate 2


title: LP-116kb_Sox2P_CM2167_rep1

description: Library name: LP-116kb_Sox2P_CM2167_rep1

tag: genotype


title: Cancer patient 15 (Discovery cohort)

description: Library name: WQ-06

value: plasma


title: male 2 sperm, cryopreserved

description: Library name: BB_2_CryoP1


title: Cancer patient 14 (Discovery cohort)

description: Library name: WQ-04


title: 4_1: Input Injury replicate 1

description: Input Injury replicate 1


title: Cancer patient 19 (Discovery cohort)

description: Library name: WQ-13


title: male 1 sperm, cryopreserved

description: Library name: BB_1_CryoP1


title: AAV_d0_4

description: Library name: Sample 19

tag: cell type


title: 3_3: Input Naïve replicate 3

description: Input Naïve replicate 3

value: Input Naïve


title: PBS_d0_4

description: Library name: Sample 18

tag: treatment

value: Saline uninjured


title: Hydrogel_3

city: Shanghai

first: Boyu

last: Xu

address: 1239 Siping Road, Shanghai, P.R. China

institute: Tongji University

description: Library name: Sample6

data_processing:
Base-calling: GATK v4.3.0.0,min base quality score 30,min read depth 10.
Data filtering: FastQC v0.11.9 for quality assessment;Trimmomatic v0.39 removed low-quality(score<20)and adapter-containing reads,parameters:sliding window 4,min quality 20,min read length 36.
Read alignment: HISAT2 v2.2.1 against GRCh38,parameters:-k 1,max intron length 100,000.
Additional processing: featureCounts v2.0.2 for quantification(-p,-B,-C);DESeq2 v1.34.0 for differential expression analysis(default parameters).
Assembly: mm10
Supplementary files format and content: FPKM, counts

organism: Oryctolagus cuniculus

source_name: Trachea

extract_protocol:
Samples are homogenized in TRIzol, centrifuged with chloroform to isolate RNA, precipitated with isopropanol, washed in ethanol, and treated with DNase I. RNA is quantified (NanoDrop), assessed for integrity (Bioanalyzer, RIN >8), and stored at -80°C. Maintain RNase-free conditions and cold handling throughout.
Purified RNA is fragmented, reverse-transcribed into double-stranded cDNA, followed by end repair and adapter ligation. PCR amplifies the library, which is then size-selected using magnetic beads. Quality control (Qubit quantification, Agilent analysis) ensures library integrity. Maintain nuclease-free conditions and precise fragment size control throughout.

value: Trachea

value: Wild Type

value: Epithelial injury + LA@PA-TA@C


title: 3_2: Input Naïve replicate 2

description: Input Naïve replicate 2


title: Cancer patient 13 (Discovery cohort)

description: Library name: WQ-03

value: Cancer patient


title: Cancer patient 12 (Discovery cohort)

description: Library name: WP-06


title: Hydrogel_2

description: Library name: Sample5


title: AAV_d14_2

description: Library name: Sample 16

tag: cell line


title: PBS_d14_2

description: Library name: Sample 17

value: PBS + 14 days post sciatic nerve crush


title: Hydrogel_1

description: Library name: Sample4


title: male 4 sperm, cryopreserved

description: Library name: BB_4_CryoP1

value: cryopreserved


title: 2_3: IP Injury replicate 3

description: IP Injury replicate 3

value: IP Injury


title: 3_1: Input Naïve replicate 1

description: Input Naïve replicate 1


title: PBS_d14_3

description: Library name: Sample 22

value: Skeletal Muscle


title: PBS_d0_3

description: Library name: Sample 20

value: Skeletal muscle cells


title: PBS_d14_1

description: Library name: Sample 15


title: Cancer patient 11 (Discovery cohort)

description: Library name: WP-05


title: AAV_d14_1

description: Library name: Sample 14

value: AAV + 14 days post sciatic nerve crush


title: Cancer patient 10 (Discovery cohort)

description: Library name: WP-04


title: Blank_2

description: Library name: Sample2

value: Epithelial deficiency


title: Cancer patient 8 (Discovery cohort)

description: Library name: WP-02


title: 2_1: IP Injury replicate 1

description: IP Injury replicate 1

value: Dissociated L4-L6 DRG


title: 2_2: IP Injury replicate 2

description: IP Injury replicate 2


title: AAV_d0_2

description: Library name: Sample 13

value: mRNA


title: Cancer patient 9 (Discovery cohort)

description: Library name: WP-03


title: Cancer patient 7 (Discovery cohort)

description: Library name: WP-01


title: Blank_3

description: Library name: Sample3


title: Cancer patient 6 (Discovery cohort)

description: Library name: EO-12


title: Blank_1

description: Library name: Sample1


title: 1_3: IP Naïve replicate 3

description: IP Naïve replicate 3

value: IP Naïve


title: AAV_d0_1

description: Library name: Sample 11

value: AAV uninjured


title: PBS_d0_2

description: Library name: Sample 12


title: Cancer patient 4 (Discovery cohort)

description: Library name: EO-10

value: plasma


title: 931-TKA-TGFb__72h

city: Freiburg im Breisgau

first: Andreas

last: Hecht

address: Stefan-Meier-Str. 17

department: Institute of Molecular Medicine and Cell Research

country: Germany

institute: Albert Ludwig University of Freiburg

data_processing:
Raw base call (BCL) files generated by Illumina sequencing were demultiplexed into FASTQ files using the cellranger mkfastq pipeline from Cell Ranger version 5.0.0
Assembly: refdata-gex-mm10-2020-A (10X genomics mouse reference)
Supplementary files format and content: .h5ad files containing raw count matrix for scRNA-seq data
Supplementary files format and content: .h5ad files containing raw count matrix for scRNA-seq data. The counts include spliced and unspliced reads

source_name: Small intestine

extract_protocol:
After mechanical disruption of 931-TKA organoids, the resulting organoid fragments were seeded in 3 mg/ml Matrigel and cultivated until they had re-formed cystic structures. Following this, organoids were treated with solvent or TGF-β1 for up to 72 h. Post-treatment, organoids were dissociated into single cells using TrypLETM Express. The resultant single organoid cells were washed once with PBS and resuspended in PBS/0.04% bovine serum albumin to enhance cell viability. Larger organoid fragments and cell debris were removed with a 30 µ MACS SmartStrainer (Miltenyi Biotec B.V. & Co. KG, Bergisch Gladbach, Germany). A BD FACSAria™ Fusion flow cytometer was then used to enrich for viable single cells.
Organoid single cells were mixed with reverse transcriptase (RT) , RT reagent, and template switch oligos, following the Chromium Next GEM Training Kit User Guide (v3.1, 10x Genomics). In brief, the cell-emzyme mixtures were combined with master mix, partitioning oil, and gel beads, then loaded into a Chromium chip and processed in the 10x Chromium controller to form gel beads in emulsion (GEMs). In GEMs, reverse transcription was performed, followed by cDNA amplification and clean-up steps. Adhering strictly to the 10x Genomics user guide, sequencing libraries were prepared, followed by SPRIselect bead cleanups. Finally, libraries from solvent and TGF-β1-treated organoid cells were pooled at a final concentration of 10 nM for sequencing.

value: Small intestine

value: Intestinal organoid

value: TGF-β1 72 h


title: 1_2: IP Naïve replicate 2

description: IP Naïve replicate 2


title: 931-TKA-TGFb__48h

value: TGF-β1 48 h


title: young_d7_2

description: Library name: Sample 9

value: 7 days post sciatic nerve crush


title: 1_1: IP Naïve replicate 1

description: IP Naïve replicate 1


title: 931-TKA-TGFb__24h

value: TGF-β1 24 h


title: 931-TKA-TGFb__0h

value: Solvent


title: Cancer patient 5 (Discovery cohort)

description: Library name: EO-11

value: Cancer patient


title: PBS_d0_1

description: Library name: Sample 10

value: Skeletal Muscle

value: Saline uninjured


title: SB-431542 treated / uncut tail (replicate 3)

city: Sheffield

first: Henry

middle: H

last: Roehl

address: Firth Court, Western Bank

country: United Kingdom

institute: University of Sheffield

description:
Library name: White-3_S17_R1_001.fastq.gz
Lexogen QuantSeq 3' FWD library kit

data_processing:
demultiplexed using the BlueBee tool (Illumina)
Assembly: GRCz11 Ensembl
Supplementary files format and content: count_matrix.tsv is the gene counts file

organism: Danio rerio

source_name: Larval tail

growth_protocol: Zebrafish embryos were raised until 72hpf at 28.5C. Fish were anesthetised in 40 μg/ml Tricaine (3-amino benzoic acidethylester) in E3. A scalpel was used to remove the end of the tail using the pigment gap as a reference

extract_protocol:
100 tails were processed for each sample. These were put in 1ml of TRI Reagent (Merck) using a plastic pestle to dissociate the tissue. Following the TRI Reagent procedure the RNA was further purified by LiCL precipitation.
Lexogen QuantSeq 3' FWD library kit

treatment_protocol: SB431542 treatments were at 50uM and were from 72-90hpf. DMSO was used as vehicle control at 0.1%.

value: Larval tail

value: Wild type

value: Uninjured SB431542 treated


title: SB-431542 treated / uncut tail (replicate 2)

description:
Library name: White-2_S16_R1_001.fastq.gz
Lexogen QuantSeq 3' FWD library kit


title: aged_d0_2

description: Library name: Sample 6

value: Skeletal muscle cells

value: aged uninjured

value: mRNA


title: Cancer patient 3 (Discovery cohort)

description: Library name: EO-09

data_processing:
Sequence reads were trimmed for adaptor sequence/low-quality sequence using Cutadapt (4.6)
PCR duplicates were removed with the BBMap tool (https://sourceforge.net/projects/bbmap/).
Random barcodes at reads end were trimmed, and low-quality reads were removed using the cutadapt tool again
Then the remaining reads were aligned to the human genome (hg38) using  bowtie2 (version 2.5.1) allowing a maximum of three mismatches.
Read count extraction and differential expression levels of exons and genes were analyzed using RADAR (0.2.4) followed by DEseq2 (1.36.0).
Cleaned reads that failed to align to the human genome were applied using Kraken2 with its standard database, which contains bacterial, archaeal, viral, and human sequences. Based on the aligned results from Kraken2, we normalized the data by calculating the reads per million (RPM). Then we performed student t-test (two tailed) to calculate the p value for the differential expression.
We identified microbial species in cfRNA samples using kraken2 (version 2.1.3)52, setting a threshold whereby the median reads of the specie must exceed 10 in at least one group (CRC or non-cancer). Following species identification, we extracted their sequences from the kraken2 reference genome. To construct a new, streamlined reference, we limited each microbial species to a maximum of ten reference genomes, minimizing redundancy Subsequently, we aligned the reads that failed to align to the human genome to this curated microbiome reference genome using bowtie2. The alignments generated by bowtie2 were applied in the calculation of the microbiome mapping ratio. Variant identification within these sequences is then achieved through the precise analysis of base composition at each genomic position, employing bam-readcount tool (https://github.com/genome/bam-readcount). The generated bam-readcount output results were parsed and analyzed to calculate the misincorporation ratio.
Assembly: hg38 (GRCh38.107)
Assembly: Kraken 2 standard database
Supplementary files format and content: abundance.host.csv|containing the abundance (raw reads), fold change, p value of various human transcirpts from cfRNA.
Supplementary files format and content: abundance.microbiome.csv|containing the RPM, fold change, p value of various microbial abundance from cfRNA.
Supplementary files format and content: mutation.microbiome.csv |containing the p value, fold change, and mutation ratio at modified sites in microbiome-dervied cfRNA.
Supplementary files format and content: mutation.host.rRNA.csv |containing the p value, fold change, and mutation ratio at human rRNA from cfRNA.
Supplementary files format and content: mutation.host.tRNA.csv |containing the p value, fold change, and mutation ratio at human tRNA from cfRNA.
Supplementary files format and content: mutation.hepG2.csv |containing mutation ratio of human tRNA from HepG2.

value: plasma


title: aged_d14_1

description: Library name: Sample 8


title: 50 weeks shell gland breeder # 58 egg in the Magnum

city: Honolulu

first: Birendra

last: Mishra

state: HI

address: 1955 East west road

department: Department of Human Nutrition Food and Animal Sciences

institute: University of Hawaii at Manoa

description:
Library name: Sample 10
Column name in Raw_Counts_All.xlsx: Aged_5

data_processing:
Miniconda 3 was used for the analysis
Adapter sequences and low-quality reads with a Phred score of less than 30 were removed with the help of the Trimmomatic function in miniconda3 (var. 27.7.3.dev31)
Clean reads were aligned with the reference genome sequence Gallus gallus (assembly bGalGal.mat.broiler.GRCg7b) in HISAT2 (var. 2.1.0).
Transcript assembly and quantification were performed with cufflinks (var. 1.5.1), available in miniconda3.
Differentially Expressed Genes (DEGs) were calculated for young broiler breeder hens about aged hens in DESeq2 (var.1.14.1) in RStudio (var.4.2.3)
Assembly: bGalGal.mat.broiler.GRCg7b
Supplementary files format and content: excel file, .xlsx file with raw count data for each samples, and DEGs in different excel sheets

organism: Gallus gallus

source_name: Magnum (part of oviduct)

extract_protocol:
RNA was extracted using TRIzol, followong standard company protocol for animal tissue
RNA concentration was measured by Qubit 3.0 using RNA BR Assay Kit (Thermo Fisher Scientific). RNA quality was determined with the Agilent Bioanalyzer Agilent 2100 Bioanalyzer (Agilent Technologies) and only samples with an RNA integrity number (RIN) of >7 were further used for RNA-sequencing. mRNA NGS libraries were prepared with NEBNext Ultra II Directional RNA Library Prep Kit (New England Biolabs) according to the manufacturer’s protocol without further modifications.

value: Magnum (part of oviduct)

value: Whole tissue

value: 50 weeks (Aged)


title: HepG2 small RNA, 1.5 ng, rep2

description: Library name: NA-08

value: hepatoblastoma

value: WT


title: SB-431542 treated / uncut tail (replicate 4)

description:
Library name: White-4_S18_R1_001.fastq.gz
Lexogen QuantSeq 3' FWD library kit


title: 50 weeks shell gland breeder # 55 egg in the Magnum

description:
Library name: Sample 9
Column name in Raw_Counts_All.xlsx: Aged_4


title: HepG2 small RNA, 1.5 ng, rep1

description: Library name: NA-07


title: aged_d7_1

description: Library name: Sample 7

value: 7 days post sciatic nerve crush


title: young_d0_3

description: Library name: Sample 4

value: young uninjured


title: DMSO treated / uncut tail (replicate 4)

description:
Library name: Pink-4_S24_R1_001
Lexogen QuantSeq 3' FWD library kit

value: Uninjured


title: SB-431542 treated / cut tail (replicate 4)

description:
Library name: Red-4_S15_R1_001
Lexogen QuantSeq 3' FWD library kit

value: Tail excision SB431542 treated


title: 50 weeks shell gland breeder # 32 egg in the Magnum

description:
Library name: Sample 6
Column name in Raw_Counts_All.xlsx: Aged_1


title: HepG2 small RNA, 5 ng, rep2

description: Library name: NA-06


title: 50 weeks shell gland breeder # 51 egg in the Magnum

description:
Library name: Sample 8
Column name in Raw_Counts_All.xlsx: Aged_3


title: DMSO treated / uncut tail (replicate 2)

description:
Library name: Pink-2_S23_R1_001
Lexogen QuantSeq 3' FWD library kit


title: Cancer patient 1 (Discovery cohort)

description: Library name: EO-07

source_name: plasma

value: Cancer patient


title: SB-431542 treated / cut tail (replicate 3)

description:
Library name: Red-3_S14_R1_001
Lexogen QuantSeq 3' FWD library kit

value: Larval tail


title: wq7

description: Library name: Sample 5

value: Skeletal Muscle


title: SB-431542 treated / cut tail (replicate 2)

description:
Library name: Red-2_S13_R1_001
Lexogen QuantSeq 3' FWD library kit

value: Wild type


title: DMSO treated / uncut tail (replicate 1)

description:
Library name: Pink-1_S22_R1_001
Lexogen QuantSeq 3' FWD library kit


title: HepG2 small RNA, 15 ng, rep2

description: Library name: NA-04


title: young_d14_308; young_d14_309 (GEX)

description:
Library name: Sample 3_GEX
young_d14_309_barcodes.tsv.gz
young_d14_309_features.tsv.gz
young_d14_309_matrix.mtx.gz
young_d14_308_barcodes.tsv.gz
young_d14_308_features.tsv.gz
young_d14_308_matrix.mtx.gz

value: Skeletal muscle cells

value: 14 days post sciatic nerve crush


title: Cancer patient 2 (Discovery cohort)

description: Library name: EO-08


title: 50 weeks shell gland breeder # 48 egg in the Magnum

description:
Library name: Sample 7
Column name in Raw_Counts_All.xlsx: Aged_2


title: 35 weeks shell gland breeder # 28 egg in the Magnum

description:
Library name: Sample 5
Column name in Raw_Counts_All.xlsx: Young_5

value: 35 weeks (Young)


title: HepG2 small RNA, 5 ng, rep1

description: Library name: NA-05


title: HepG2 small RNA, 15 ng, rep1

description: Library name: NA-03


title: 35 weeks shell gland breeder # 8 egg in the Magnum

description:
Library name: Sample 2
Column name in Raw_Counts_All.xlsx: Young_2


title: young_d7_306; young_d7_307 (GEX)

description:
Library name: Sample 2_GEX
young_d7_307_barcodes.tsv.gz
young_d7_307_features.tsv.gz
young_d7_307_matrix.mtx.gz
young_d7_306_barcodes.tsv.gz
young_d7_306_features.tsv.gz
young_d7_306_matrix.mtx.gz

value: mRNA


title: 35 weeks shell gland breeder # 26 egg in the Magnum

description:
Library name: Sample 4
Column name in Raw_Counts_All.xlsx: Young_4

value: Magnum (part of oviduct)


title: HepG2 small RNA, 50 ng, rep2

description: Library name: NA-02


title: 35 weeks shell gland breeder # 24 egg in the Magnum

description:
Library name: Sample 3
Column name in Raw_Counts_All.xlsx: Young_3

value: Whole tissue


title: young_301; young_308 (GEX)

description:
Library name: Sample 1_GEX
young_308_barcodes.tsv.gz
young_308_features.tsv.gz
young_308_matrix.mtx.gz
young_301_barcodes.tsv.gz
young_301_features.tsv.gz
young_301_matrix.mtx.gz


title: DMSO treated / cut tail (replicate 4)

description:
Library name: Orange-4_S21_R1_001
Lexogen QuantSeq 3' FWD library kit

value: Tail excision


title: 35 weeks shell gland breeder # 7 egg in the Magnum

description:
Library name: Sample 1
Column name in Raw_Counts_All.xlsx: Young_1


title: DMSO treated / cut tail (replicate 1)

description:
Library name: Orange-1_S19_R1_001
Lexogen QuantSeq 3' FWD library kit


title: young_d7_306; young_d7_307 (CMO)

description:
Library name: Sample 2_CMO
young_d7_307_barcodes.tsv.gz
young_d7_307_features.tsv.gz
young_d7_307_matrix.mtx.gz
young_d7_306_barcodes.tsv.gz
young_d7_306_features.tsv.gz
young_d7_306_matrix.mtx.gz

value: CMO

tag: cmo


title: young_301; young_308 (CMO)

description:
Library name: Sample 1_CMO
young_308_barcodes.tsv.gz
young_308_features.tsv.gz
young_308_matrix.mtx.gz
young_301_barcodes.tsv.gz
young_301_features.tsv.gz
young_301_matrix.mtx.gz


title: HepG2 small RNA, 50 ng, rep1

description: Library name: NA-01

value: hepatoblastoma

value: WT


title: DMSO treated / cut tail (replicate 3)

description:
Library name: Orange-3_S20_R1_001
Lexogen QuantSeq 3' FWD library kit


title: young_d14_308; young_d14_309 (CMO)

description:
Library name: Sample 3_CMO
young_d14_309_barcodes.tsv.gz
young_d14_309_features.tsv.gz
young_d14_309_matrix.mtx.gz
young_d14_308_barcodes.tsv.gz
young_d14_308_features.tsv.gz
young_d14_308_matrix.mtx.gz


title: AM403 infected

status: Public on May 15 2025

type: RNA

city: Dublin

first: Aaron

last: Walsh

address: Trinity Translational Medicine Institute

department: Department of Clinical Medicine

country: Ireland

institute: Trinity College Dublin

description: nCounter Metabolic Pathways Panel [NS_Hs_Metabolism_v1.0]

hyb_protocol: NanoString nCounter Metabolic Pathways Panel

data_processing: Data were analysed using the NanoString nSolver Data Analysis Software

source_name: Human alveolar macrophages

label_protocol: NanoString

extract_protocol: AM were infected with live virulent Mtb H37Rv for 24 hours (hr) using a multiplicity of infection of 1-10 bacilli per cell, as previously described. AM were then lysed, and total RNA was collected and purified using silica-membrane column centrifugation.

tag: subject id

tag: group

value: infected

tag: smoking

value: ex-smoker

value: Human alveolar macrophages


title: patient23

first: Yidong

last: Zhang

state: Bijing

address: No 1. Shuaifuyuan, Dongcheng District

institute: Peking Union Medical College Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College

description:
Library name: Sample 23
patient23

data_processing:
fastq files were aligned to version 37 of human reference genome GRCh37 using STAR 2.7.6a.
BAM files resulting from the alignments were summarized to genes using the STAR 2.7.6a algorithm
Expression data was normalized using the VOOM function in the Limma package in R
Assembly: version 37 of human reference genome GRCh37
Supplementary files format and content: tab-delimited text file with reads counts

source_name: cervical cancer surgical speciman

extract_protocol:
RNA was isolated using the ReliaPrep FFPE gDNA Miniprep System (Promega) and processed as per manufacturer’s instructions.
libraries were prepared using the Hieff NGS® Ultima Dual-mode mRNA Library Prep Kit for Illumina® (Cat#12301) Kit, according to the manufacturer’s instructions.

value: cervical cancer surgical speciman


title: patient22

description:
Library name: Sample 22
patient22


title: AM403 uninfected

scan_protocol: NanoString nCounter Metabolic Pathways Panel

value: uninfected


title: AM365 infected


title: AM364 infected


title: AM364 uninfected


title: patient21

description:
Library name: Sample 21
patient21


title: AM365 uninfected


title: AM404 uninfected

value: never-smoker


title: AM404 infected


title: patient20

description:
Library name: Sample 20
patient20


title: patient19

description:
Library name: Sample 19
patient19


title: AM362 infected


title: AM366 uninfected


title: AM362 uninfected


title: patient18

description:
Library name: Sample 18
patient18


title: patient17

description:
Library name: Sample 17
patient17


title: patient16

description:
Library name: Sample 16
patient16


title: patient15

description:
Library name: Sample 15
patient15


title: patient12

description:
Library name: Sample 12
patient12


title: patient10

description:
Library name: Sample 10
patient10


title: patient14

description:
Library name: Sample 14
patient14


title: patient13

description:
Library name: Sample 13
patient13


title: patient9

description:
Library name: Sample 9
patient9


title: patient7

description:
Library name: Sample 7
patient7


title: patient11

description:
Library name: Sample 11
patient11


title: patient4

description:
Library name: Sample 4
patient4


title: patient1

description:
Library name: Sample 1
patient1


title: patient6

description:
Library name: Sample 6
patient6


title: patient5

description:
Library name: Sample 5
patient5


title: ko_emt2

city: New York

first: Samie

last: Jaffrey

state: New York

address: 1300 York Ave

institute: Weill Cornell Medicine

description: Library name: ko_emt2

data_processing:
Library quality was first analyzed using MultiQC 1.14.  Adaptors were trimmed using TrimGalore-0.6.9 with --stringency 3 and library quality was again assessed using MultiQC 1.14.  The data was then analyzed using the slamdunk all function of the analytical suite SLAM-DUNK 0.4.3 with the default parameters for mapping, quality filtering, and single nucleotide polymorphism identification.  The reference genome hg38 and mm10 was used for mapping and the subsequent analysis. PCR duplicates in the quality filtered reads were collapsed using alleyoop collapse function of SLAM-DUNK 0.4.3.  The 4sU conversion was further assessed using alleyoop rates, alleyoop tccontext, and alleyoop snpeval functions with the default parameters.  alleyoop utrrates was used with a bed file of all exons of each mRNA to analyzed 4sU conversion rates per mRNA.  The quality filtered and PCR duplicate-collapsed mapped reads were further analyzed using GRAND-SLAM 2.0.7b with the default parameters to quantify read counts and perform statistical analysis to infer the ratio of transcripts for each gene that originate from pre-4sU labeling and post labeling.  The results from GRAND-SLAM 2.0.7b was used to calculate mRNA synthesis rate and half-life using grandR 0.2.2.
Assembly: mm10
Supplementary files format and content: The processed data file contains the log2 fold change on half-life, the lower/upper credible interval boundaries of the half-life log2 fold change, and the signed ROPE probability.

source_name: mESC

growth_protocol: mESC cells were grown in KnockOut DMEM (ThermoFisher, 10829018) supplemented with 15% heat-inactivated FBS, 10 U/mL Penicillin-Streptomycin (ThermoFisher, 15140122), 1x GlutaMax (ThermoFisher, 35050061), 500 μM β-mercaptoethanol (ThermoFisher, 21985023), 1x Nonessential amino acids (ThermoFisher, 11140076), 103 U/mL LIF (MilliporeSigma, ESG1107), 3 μM CHIR99021 (ThermoFisher, 501013679), and 1 μM PD0325901 (ThermoFisher, 501013147). Cells were plated on plates coated with EmbryoMax™ 0.1% gelatin solution (MilliporeSigma, ES-006-B) and passaged daily using TrypLE Express (ThermoFisher, 12604039) according to the manufacturer’s instructions.

extract_protocol:
Total RNA was collected using Qiagen RNeasy Mini kit RLT buffer (Qiagen, 74104) supplemented with 1% 𝛽-mercaptoethanol (BME).  Cells were lysed by passing through a 25 1/2-gauge needle, and RNA was isolated following the manufacturer’s instruction with RPE buffer supplemented with 1% BME.
10 µg total RNA was treated with 2,2,2-trifluoroethylamine (TFEE) and sodium periodate (NaIO4) at 45 °C for 1 h to convert 4sU to N4-trifluoroethylcytosine.  RNA was then purified twice using RNAClean beads (Beckman Coulter, A63987). 1 µg of treated RNA was used for RNA-seq library preparation using NEBNext® rRNA Depletion Kit (NEB, E6310X) and NEBNext® Ultra™ II Directional RNA Library Prep Kit (NEB, E7760L).  Library quality was assessed on Agilent TapeStation4200 using D1000 ScreenTape (Agilent, 5067-5584).  Library was quantified on Qubit4 Fluorometer using Qubit 1X dsDNA HS Assay Kit (ThermoFisher, Q33231).

treatment_protocol: Cells were incubated treated with 250 µM 4-thiouridine (4sU, SigmaAldrich, T4509) for 3 hr.

value: mESC

value: wildtype


title: ko_emt3

description: Library name: ko_emt3


title: ko_ctrl2

description: Library name: ko_ctrl2


title: AM366 infected


title: ko_ctrl1

description: Library name: ko_ctrl1


title: patient3

description:
Library name: Sample 3
patient3


title: patient2

description:
Library name: Sample 2
patient2


title: wt_ctrl2

description: Library name: wt_ctrl2


title: wt_ctrl3

description: Library name: wt_ctrl3


title: wt_ctrl1

description: Library name: wt_ctrl1


title: patient8

description:
Library name: Sample 8
patient8


title: CropSeq Colitis 2, Sample 3, ADT

city: Hamburg

first: Malte

last: Hellmig

state: Hamburg

address: Martinistraße 52

department: III. Department of Medicine

institute: University Medical Center Hamburg-Eppendorf

description:
Library name: 23_2-Th17-2-ADT
10X Genomics scRNA-seq using Chromium Single Cell 3' v7

data_processing:
10x Genomics raw sequencing data were processed using CellRanger software (version 3.1 and 7.1, 10x Genomics, Pleasanton, CA).
Assembly: GRCh38
Assembly: mm10
Supplementary files format and content: Cellranger output: Tab seperated values files and Matrix table with raw gene counts
Supplementary files format and content: Cellranger output: Character seperated values file of TCR contig annotation
Supplementary files format and content: Cellranger output: Filtered feature-barcode matrices HDF5. JSON object where each protospacer is associated with a list of barcodes.
Library strategy: CITE-seq

molecule: protein

source_name: intestine

extract_protocol:
Kidneys were incubated at 37 °C for 40 min in RPMI 1640 Medium (Thermo Fisher) containing 10% heat inactivated FBS, 1% P/S, 0.4 µg/ml Collagenase D (Roche, Basel, Switzerland) and 0.2 µg/ml DNAse (Roche). Subsequently, tissue was dissociated using the GentleMACS Dissociator and C-Tubes (both Miltenyi Biotec) and leukocytes were isolated using a gradient centrifugation with 37 % Percoll (GE Healthcare). After the centrifugation, cells were washed with DPBS (Gibco). CD4+ T cells were enriched using the EasySep Mouse CD4+ T cell Isolation Kit according to manufacturer’s instructions (Stemcell Technologies, Vancouver, Canada). Enriched cells were sorted by Fluorescence-activated cell sorting (FACS) for CD4+ T cells, GFP signal and eBFP2 signal. For isolation of intestinal lymphocytes, the small intestine was obtained and cut into 1 cm pieces. We separated lamina propria lymphocytes (LPLs) from intraepithelial lymphocytes (IELs) by incubation with 2 mM DTT at 37°C for 30 min and subsequent filtering through a 100 µm cell strainer (Falcon). The IELs in the supernatant were discarded. The remaining parts were transferred into 50 ml tubes (Falcon, Thermo Fischer), containing DPBS, calcium, magnesium (Gibco), washed by rigorous shaking and repeatedly filtered through a 100 µm cell strainer. Further, the pieces were digested with collagenase D (Roche) and DNAse (Roche) at 37°C for 45 min while rotating in a MACS-Mix tube rotator (Miltenyi Biotec). Subsequently, the small intestines were minced in a MACS cell separator (C-tubes, program “intestine 01”) and filtered through a 70 µm cell strainer (Falcon) until the tissue turns white. The cell strainer was washed with RPMI-medium. Lastly, gradient centrifugation with 37% Percoll (GE Healthcare) was performed. Cells were sorted by Fluorescence-activated cell sorting (FACS) for CD4+ T cells, GFP signal and eBFP2 signal.
All steps were performed according to standard protocol of the Chromium Single Cell 3' v3 kit, Chromium  Next GEM Single Cell 3' v3.1

value: intestine

value: T cells

value: experimental colitis

value: ADT

tag: adt

value: Features defined in CropSeq_feature_reference.csv


title: CropSeq Colitis 2, Sample 2, ADT

description:
Library name: 23_2-Th17-1-ADT
10X Genomics scRNA-seq using Chromium Single Cell 3' v7


title: CropSeq Colitis 2, Sample 1, ADT

description:
Library name: 23_2-Control-ADT
10X Genomics scRNA-seq using Chromium Single Cell 3' v7


title: CropSeq Colitis 2, Sample 1, GEX

description:
Library name: 23_2-Control-GEX
10X Genomics scRNA-seq using Chromium Single Cell 3' v7

data_processing:
10x Genomics raw sequencing data were processed using CellRanger software (version 3.1 and 7.1, 10x Genomics, Pleasanton, CA).
Assembly: GRCh38
Assembly: mm10
Supplementary files format and content: Cellranger output: Tab seperated values files and Matrix table with raw gene counts
Supplementary files format and content: Cellranger output: Character seperated values file of TCR contig annotation
Supplementary files format and content: Cellranger output: Filtered feature-barcode matrices HDF5. JSON object where each protospacer is associated with a list of barcodes.

value: mRNA


title: CropSeq NTN 2, Sample 4, CRISPR

description:
Library name: 23_1-Th17-3-CRISPR
10X Genomics scRNA-seq using Chromium Single Cell 3' v7

source_name: kidney

value: kidney

value: crescentic glomerulonephritis

value: CRISPR

tag: crispr


title: CropSeq NTN 2, Sample 4, ADT

description:
Library name: 23_1-Th17-3-ADT
10X Genomics scRNA-seq using Chromium Single Cell 3' v7


title: CropSeq NTN 2, Sample 3, ADT

description:
Library name: 23_1-Th17-2-ADT
10X Genomics scRNA-seq using Chromium Single Cell 3' v7


title: wt_emt2

description: Library name: wt_emt2


title: ko_emt1

description: Library name: ko_emt1


title: CropSeq NTN 2, Sample 2, GEX

description:
Library name: 23_1-Th17-1-GEX
10X Genomics scRNA-seq using Chromium Single Cell 3' v7


title: CropSeq Colitis 2, Sample 3, GEX

description:
Library name: 23_2-Th17-2-GEX
10X Genomics scRNA-seq using Chromium Single Cell 3' v7