title: INP - mCD8-RFP scRNAseq

data_processing:
Quality control for all raw *.fastq.gz-files was performed with FastQC (v0.11.5).
Reads were mapped to the Drosophila dm6 genome assembly with bowtie2 (v2.3.4.1).
Read counts per 500 bp-bin were acquired by 'bamCoverage' based on *.bam-files of individual samples including a read extension to 150 bp (https://deeptools.readthedocs.io/en/develop/content/tools/bamCoverage.html).
Reads were assigned to GATC-fragment bins based on 'GATC fragment'-file generated with 'gatc.track.maker.pl' (https://github.com/AHBrand-Lab/DamID_scripts).
All pairwise 'NanoDam/TaDa-vs-DamOnly'-comparisons were quantile normalized to each other via 'quant_norm_bedgraph.pl' (https://github.com/AHBrand-Lab/DamID_scripts).
Quantile normalized files were averaged with 'average_tracks.pl' (https://github.com/AHBrand-Lab/DamID_scripts).
Bedgraph files were converted into the bigwig-format with 'bedGraphToBigwig' (https://www.encodeproject.org/software/bedgraphtobigwig/).
Peak calling was conducted using MACS2 ('--broad') based on *.bam files from pairs of each one NanoDam/TaDa- and one DamOnly-sample.
All steps were performed for all files in an automated manner via 'damMer.py', 'damMer_tracks.py' and 'damMer_peaks.py' based on the 'damidseq_pipeline_vR.1' and the slurm workload manager (v15.08.13; https://github.com/AHBrand-Lab/NanoDam_analysis).
All single cell RNA sequencing files were mapped and count matrices generated by Cell Ranger (v2.1.1.).
Genome_build: dm6
Supplementary_files_format_and_content: *.sort.bam.bin500.ext150.bgr'-files (bedgraph-format): Raw read counts per 500 bp-bin after extension to 150 bp.
Supplementary_files_format_and_content: *_vs_*.bedgraph'-files (bedgraph-format): Normalized, averaged scores across all pairwise comparisons (i.e., DichaeteGal4_*; OgreGal4_*) or scores for individual comparisons normalized across all comparisons (i.e., Cph_*).
Supplementary_files_format_and_content: *_vs_*.bw'-files (bigwig-format): Normalized, averaged scores across all pairwise comparisons (i.e., DichaeteGal4_*; OgreGal4_*) or scores for individual comparisons normalized across all comparisons (i.e., Cph_*).
Supplementary_files_format_and_content: *_vs_*.broadPeak'-files (bed-format): List of broad peaks as called by MACS2 on individual, pairwise comparisons.
Supplementary_files_format_and_content: *.mergePeak' (bed-format): Peakset merged across peaks of all individual, pairwise comparisons that satisfied a particular FDR threshold.
Supplementary_files_format_and_content: *.reproPeak' (bedgraph-format): '*.mergePeak' (bed-format): Reproducible peakset merged across peaks of all individual, pairwise comparisons that satisfied a particular FDR threshold and that occur in at least 50% of all comparisons.
Supplementary_files_format_and_content: *.tsv' (tsv-format): Lists of barcodes and gene identifiers accompanying single cell count matrices.
Supplementary_files_format_and_content: *.mtx' (mex-format): Single cell count matrices.

source_name: Type 2 Neural Stem Cell-derived Intermediate Progenitors & offspring - 3rd instar larval brain

growth_protocol: Embryos collected at 25°C on yeasted apple juice plates; Larvae reared at 25°C on yeasted food plates until wandering 3rd instar

extract_protocol:
Single cells extracted according to Harzer, H et al., 2013 with PBS instead of Schneider's medium
For all DamID-samples, genomic DNA was extracted with the QiAmp DNA Micro kit, digested with DpnI, DamID-adaptors were ligated followed by DpnII-digestion and PCR-amplification (Marshall, O et al., 2016, Nat Protoc).
For all DamID-libraries, the amplified products were sonicated to average size of 300 bp, DamID-adaptors removed by AlwI-digestion, end-repaired, A-tailed, followed by ligation of Sequencing-adaptors and PCR-amplification (see Marshall, O et al., 2016, Nat Protoc).
Single cell RNAseq libraries were generated with the 10xGenomics 'SC3Pv2'-chemistry kit.

value: Wandering 3rd instar larval brain

tag: expression conditions

value: Dichaete-Gal4-driven expression throughout larval development

tag: cell type

tag: dam complex


title: mTGN_RFSL1_3_1

status: Public on Aug 03 2022

type: SRA

city: Kaifeng, Henan Province

first: Yanqing

last: Li

address: Jinming Avenue

country: China

institute: Henan University

library_source: transcriptomic

data_processing:
Base calling and the quality score of each base are calculated based on the phred-33 quality score standard by the sub-Pixel Registration algorithm according to the optical signal intensity of each lane
This project uses the filtering software SOAPnuke (Versionv1.5.2) developed by BGI independently for filtering. SOAPnuke   Official Websitehttps://github.com/BGI-flexlab/SOAPnuke The specific steps are as follows: 1) Remove the reads containing the adaptor (adaptor pollution); 2) Remove reads whose N content is greater than 5%; 3) Remove low-quality reads (we define reads with bases with a quality score less than 10 as the proportion of total bases in the reads that are greater than 20% as low-quality reads).
Hierarchical Indexing for Spliced Alignment of Transcripts (HISTAT) is software for mapping RNA-seq reads. HISAT will use the global FM index to anchor the position of partial sequences in each read on the genome; then, use the partial genome indexes of these alignment positions to align the remaining sequences of each read to extend the alignment area. Software information:HISAT2 (Version：v2.0.4) ,Official Website：http://www.ccb.jhu.edu/software/hisat
Bowtie2 (v2.2.5) was used to compare clean reads to reference sequences for gene alignment with -q --phred64 --sensitive --dpad 0 --gbar 99999999 --mp 1,1 --np 1 --score-min L,0,-0.1 -p 16 -k 200. And then RSEM (v1.2.8) was used to calculate gene and transcript expression levels with -p 8 --forward-prob 0.5 --paired-end.
Genome_build: GCF_000001635.26_GRCm38.p6 for Mus musculus
Supplementary_files_format_and_content: Excel files include FPKM values, Reads Count for each Sample

molecule: total RNA

organism: Mus musculus

source_name: mouse trigeminal ganglion neurons

growth_protocol: Mouse dorsal root ganglia (mDRGs) or trigeminal neurons (mTGNs) were isolated from postnatal d5 C57BL/6 mice after the mice after being deeply anesthetized by i.p. injection of a mixture of 0.2% xylazine, 1% ketamine, and 0.9% NaCl. The harvested ganglia were incubated with Hank’s balanced salt solution (HBSS) using collagenase I (10 mg/mL, Sigma) and dispase II (5 mg/mL, Sigma) for 30 minutes. Digests were washed with DMEM and the cell suspension was filtered through a 100-μm cell strainer and cultured in 24-well plates pre-coated with poly-L-lysine (Sigma) and laminin (Sigma) in medium consisting DMEM supplemented with 5% (v/v) FBS (Sigma), 100 U/ml penicillin, 100 μg/ml streptomycin in the presence of cytosine β-d-arabinofuranoside (Sigma) 10 µM, 1xB27, and nerve growth factor 50 ng/ml (Sigma) for 7 days

extract_protocol:
Remove the upper medium and wash it twice with cold PBS, frozen on ice, and RNA was harvested using Trizol reagent.The mix was centrifuged for 5 minutes at 12,000×g at 4°C, then the supernatant was transferred into a new EP tube with 0.3 mL chloroform/isoamyl alcohol (24:1). The mix was shacked vigorously for 15s, and then centrifuged at 12,000×g for 10 minutes at 4°C. After centrifugation, the upper aqueous phase where RNA remained was transferred into a new tube with equal volume of supernatant of isopropyl alcohol, then centrifuged at 13,600 rpm for 20 minutes at 4°C. After deserting the supernatant, the RNA pellet was washed twice with 1 mL 75% ethanol, then the mix was centrifuged at 13,600 rpm for 3 minutes at 4°C to collect residual ethanol, followed by the pellet air dry for 5-10 minutes in the biosafety cabinet. Finally, 25µL~100µL of DEPC-treated water was added to dissolve the RNA. Subsequently, total RNA was qualified and quantified using a Nano Drop and Agilent 2100 bioanalyzer (Thermo Fisher Scientific, MA, USA).
Oligo(dT)-attached magnetic beads were used to purified mRNA. Purified mRNA was fragmented into small pieces with fragment buffer at appropriate temperature. Then First-strand cDNA was generated using random hexamer-primed reverse transcription, followed by a second-strand cDNA synthesis. afterwards, A-Tailing Mix and RNA Index Adapters were added by incubating to end repair. The cDNA fragments obtained from previous step were amplified by PCR, and products were purified by Ampure XP Beads, then dissolved in EB solution. The product was validated on the Agilent Technologies 2100 bioanalyzer for quality control. The double stranded PCR products from previous step were heated denatured and circularized by the splint oligo sequence to get the final library. The single strand circle DNA (ssCir DNA) was formatted as the final library. The final library was amplified with phi29 to make DNA nanoball (DNB) which had more than 300 copies of one molecular, DNBs were loaded into the patterned nanoarray and single end 50 bases reads were generated on DNBSeq platform (BGI-Shenzhen, China)

treatment_protocol: mTGNs were treated with Pam3CSK4 (1 μg/ml)  or FSL-1 (1 μg/ml)  for 6h;mDRGs were treated with serpinE1(20μg/ml)  for 6h;

value: neurons

tag: cell subtype

value: trigeminal ganglion

tag: treatment

value: FSL-1 (1 microg/ml)  treated


title: EARLI_10741_Sepsisperiph

city: San Francisco

first: Charles

last: Langelier

state: CA

address: HSE 401, 513 Parnassus Ave

department: Medicine

country: USA

institute: University of California San Francisco

data_processing:
Alignment against human genome- star
Fitering to retain samples with 50,000 gene counts
Differential expression with R package DESeq2
Genome_build: HG-38, ENSEMBL v. 99
Supplementary_files_format_and_content: .csv files with genecounts

organism: Homo sapiens

source_name: Plasma

growth_protocol: Clinical plasma specimens

extract_protocol:
Zymo DNA/RNA magbead
NEBnext with Qiagen Fastselect rRNA depletion
Random hexamer RNAseq

treatment_protocol: DNAse

tag: disease state

value: Sepsisperiph

tag: tissue

value: Plasma

tag: gender

value: Female

tag: age

tag: race

value: Asian

tag: ethnicity


title: 1095

city: Memphis

first: Gang

last: Wu

address: 262 Danny Thomas Place

institute: St Jude Children's Research Hospital

data_processing:
Standed mRNA sequencing data were processed by  the internal AutoMapper pipeline.
First the raw reads were firs trimmed (Trim-Galore version 0.60),  mapped to human genome assembly (GRCh37-lite) (STAR v2.7)
and then the gene level values were quantified (RSEM v1.31) based on GENCODE annotation (r19) .
Assembly: hg19
Supplementary files format and content: gene-level RSEM raw count, TPM, FPKM txt files

source_name: Blood

extract_protocol:
Lymphoblasts were purified from bone marrow or peripheral blood specimens using the Ficoll-Paque centrifugation method and RNA was extracted using NucleoZol reagent (MACHEREY-NAGEL, Cat#740404)
Libraries were prepared from 1~1.5 ug total RNA using TruSeq Stranded mRNA preparation kit and mRNA was enriched using oligo-dT capture.
Stranded mRNA

value: Blood

value: B-cell acute lymphoblastic leukemia


title: mTGN_RFSL1_2_1


title: INP - Dichaete-GFP mCD8-RFP scRNAseq

city: Cambridge

first: Andrea

last: Brand

address: Tennis Court Rd

country: United Kingdom

institute: The Gurdon Institute

organism: Drosophila melanogaster

value: Type 2 Neural Stem Cell-derived Intermediate Progenitors & offspring


title: EARLI_10740_Sepsisperiph

value: Male

value: African American


title: 1081


title: EARLI_10456_Sepsisperiph


title: 1079


title: 1073


title: mTGN_ctrl_2_1

value: untreated


title: mTGN_ctrl_3_1


title: 574_G

city: Corvallis

first: Massimo

last: Bionaz

state: OR

address: Weniger Hall

department: Animal Sciences

institute: Oregon State University

data_processing:
Quality control was assayed using MultiQC v1.8 [158] (https://multiqc.info/). Reads were then trimmed based on PHRED score and adapter presence using TRIMMOMATIC v0.39 [159](https://github.com/usadellab/Trimmomatic) with arguments LEADING:5 TRAILING:5 SLIDINGWINDOW:4:5 MINLEN:3
A genome index was generated using the ARS-UCD1.2 Bos taurus genome (http://ftp.ensembl.org/pub/release-104/fasta/bos_taurus/dna/) and the ARS-UCD1.2.104 annotation (http://ftp.ensembl.org/pub/release-104/gtf/bos_taurus/), using the genomeGenerate function of STAR v2.7.1 [160](https://github.com/alexdobin/STAR).
). Trimmed reads were aligned against the reference genomic index using STAR, and the average overall alignment rate was 85.46%.
Aligned .sam files were sorted and converted to .bam using samtools v1.0 [161](https://github.com/samtools/samtools), and gene count matrices were generated using stringtie v 2.0 (https://ccb.jhu.edu/software/stringtie/).
Differential expression was determined using the DESeq2 package, v1.30.1 [162](https://bioconductor.org/packages/release/bioc/html/DESeq2.html) in R v3.9, after filtering for low counts (any transcript with raw count ≤ 4). Three contrasts were generated (PPARA agonist vs CTR, PPARD agonist vs CTR, and PPARG agonist vs CTR) and DEG were considered significant with FDR ≤ 0.2.
Genome_build: ARS-UCD1.2 Bos taurus genome
Supplementary_files_format_and_content: liverslices_counts.xlsx   contains raw counts for all samples

organism: Bos taurus

source_name: Precision-cut bovine liver slices

growth_protocol: PCLS were prepared following the protocol developed by De Graaf and collaborators [155], with few modifications. Briefly, liver tissue samples were transported to the laboratory in ice-cold KHB; upon arrival, they were immediately embedded in low gelling ultrapure agarose (16500-100, Invitrogen, CA, USA) inside a 8 mm mold-plunger assembly (MD2200, tissue embedding unit, Alabama Research & Development, AL, USA). The plunger with the embedded tissue was then transferred to a Krumdieck Tissue Slicer (MD1000-A1, Alabama Research & Development, AL, USA), pre-filled with ice-cold KHB; slice thickness was set at 280-300 µm, with a cycle speed of ~35 and using the “intermittent blade mode”. Slices were then collected and placed on a new tissue culture dish, prefilled with a minimum amount of cold KHB to prevent dehydration. A total of 36 PCLS were selected for each animal at each timepoint, evaluating visually to identify those with the best-defined shape, and without holes or patent morphological irregularities. The 36 PCLS were thus transferred to three 12-well culture plates (665180, Greiner Bio One, NC, USA) and the treatment were supplied in duplicates. In Plates 1 and 2, PCLS were cultured in William’s Medium E (WME, A1217601, Gibco, Thermo Fisher Scientific, MA, USA), supplemented with GlutaMAX (35050-061, Gibco, NY, USA), 14 mM of D-glucose monohydrate (0643-1KG, VWR, OH, USA), and 50 µg/mL of gentamycin (15750060, Life Technologies, OR, USA).

extract_protocol:
PCLS from Plates 2 and 3 were collected after incubation, and transferred to separate screw-cap vials (490003-520, VWR, PA, USA), pre-filled with 600 µL of ice-cold TRIzol reagent (15596026, Thermo Scientific, MA, USA) and two 3.2 mm beads, homogenized using a Geno/Grinder Automated Tissue Homogenizer (2010-115, SPEX SamplePrep, NJ, USA; courtesy of the Department of Crop and Soil Sciences, Oregon State University, OR, USA), disrupting the tissue in short burst (45 seconds) at 1500 rpm, followed by incubation on ice for 3 minutes; the disruption-incubation cycle was repeated up to three times, or until no tissue pieces were visible within the tubes. Immediately after disruption, 120 µL of pre-chilled chloroform was added the tube, and the samples were mixed by inverting the tube, and incubated on ice for 5 minutes. After incubation, the samples were transferred to a new 1.7 mL microcentrifuge tube, and centrifuged a 4 C for 15 minutes, 15,000 x g. The upper-phase supernatant (~200 µL) was collected and RNA was purified using a Mag-MAX-96 Total RNA Isolation Kit (AM1830, Invitrogen, MA, USA) following the manufacturer’s instructions, with minor modifications: briefly, 100 µL of each sample was transferred to the first row (A) of a 96-Well DeepWell Storage Plate (260251, Thermo Scientific, MA, USA). Rows B-F contained reagents supplied with the kit: 20 µL of the magnetic beads mix (row B), 150 µL of wash solution 1 (row C), 150 µL of wash solution 2 (row D), and 50 µL of DNAse-RNAse free water (VWRL0201-0500, VWR, PA, USA) in rows E and F. A suitable protocol was then generated to mimic the manufacturer’s protocol with a KingFisher Duo Purification System (5400110, Thermo Scientific, MA, USA). Eluted RNA was measured using a SpectraDrop Micro-Volume Microplate in a SpectraMax plus 384 spectrophotomoter (89212-396, Molecular Devices, CA, USA). Average 260/230 and 260/280 ratios were 2.06 ± 0.24 and 1.69 ± 0.27, respectively. RNA integrity was assessed by the Center for Genome Research and Bioinformatics at Oregon State University using an Agilent Bioanalyzer 2100 (G2939BA, Agilent, CA, USA). For one animal, +10 DIM PCLS were excluded from the analysis, as their RIN was below 3; unsurprisingly, these samples clustered together in the principal component analysis of RNAseq results and were very dissimilar from the other animals, which further justified their removal; with that sample removed, resulting RNA integrity numbers were 7.26 ± 0.64.
PCLS from postpartum animals treated with the three isotype-specific PPAR agonists, as well as the control group, were sent to the Center for Genome Research and Bioinformatics at Oregon State University for high-throughput sequencing. Library construction was obtained using a QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina (015.96, Lexogen, NH, USA). Sequencing was performed on an Illumina HiSeq3000 platform, at 60 samples/lane. The raw reads have been deposited

treatment_protocol: ). Within the plate, PCLS were treated with 100 µM of the PPARα agonist WY-14643 (CATNO,PRODUCT,PLACE), 50 µM of the PPARδ agonist GW-501516 (ALX-420-032-M005, Enzo, NY, USA), or 100 µM of the PPARγ agonist rosiglitazone (R0106, TCI, OR, USA); 200 µM of palmitic acid (100905, MP Biomedicals, CA, USA) or 100 µM of serum NEFA, isolated via solid phase extraction as previously reported [145]. WY-14643 concentration was selected based on prior reports [156]; additionally, according to our findings in an immortalized model of bovine liver [145], the dose-dependent response to PPARα and PPARγ is similar (hence the 100 µM concentration of both WY and rosiglitazone), while PPARδ modulation was about twice as sensitive (hence the 50 µM dose for GW-501516). Additionally, peak PPAR activation for palmitic acid was achieved at 200 µM. The concentration of NEFA was chose to mimic the physiological concentration of NEFA in the lactating dairy cow. In Plate 3, PCLS from each cow were cultured in blood serum isolated from the same cow on the day of the liver biopsy; PPAR antagonists were added at 50 µM in duplicates to the wells: for PPARα, GW-6471 (9453, CAS# 880635-03-0, BioVision incorporated, CA, USA); for PPARδ, GSK-3787 (3961/10, CAS# 188591-46-0, Tocris, Bio-Techne Corporation, MN, USA); for PPARγ, GW-9662 (70785, CAS# 22978-25-2, Cayman Chemicals, MI, USA). Additionally, palmitic acid and serum NEFA were also supplemented with doses as in Plates 1 and 2. All treatments were adjusted for the vehicle (DMSO, D2438, Millipore Sigma, MO, USA) at a volume of 0.8%. Plates were placed in a modular incubator chamber (MIC-101, Billups-Rothenberg, CA, USA), clamped shut, and flushed with carbogen (95 % 02, 5% CO2) for 10 minutes. The modular chamber was placed inside a cell culture incubator, and atop a benchtop orbital shaker located inside of the incubator. The slices were incubated for 18 hours at 37 C, ~100 rpm.

value: Liver

value: PPARG agonist


title: SKMEL5HYP2

city: Miami

first: Anthony

last: Griswold

state: FL

address: 1501 NW 10th Ave, BRB 318

institute: University of Miami

data_processing:
FASTQ files were created with the Illumina supplied scripts in the BCL2FASTQ software (v2.20)
Illumina adapters trimmed with Trim Galore! V0.6.1
FASTQs aligned to the GRCh38 reference genome with STAR v2.5.0a using default parameters
--quantMode GeneCounts option in STAR used to count genes against the GENCODE v35 GTF annotation file
Genome_build: GRCh38
Supplementary_files_format_and_content: Processed file is a tab delimited matrix file with the first column representing the Ensembl gene name and subsequent columns raw gene counts generarted by --quantMode GeneCounts option in STAR. A read is counted if it overlaps (1bp or more) with an exon of only one gene on the appropriate strand.

source_name: Metastatic melanoma cell line

growth_protocol: Normoxic cells were cultured under atmospheric oxygen tension. Hypoxic samples were cultured under 1% oxygen tension.

extract_protocol:
RNA was extracted using QUIAGEN Rneasy Mini Kit
Libraries were constructed using 600ng of total RNA with the NuGen Universal Plus mRNA protocol.

treatment_protocol: Hypoxic growth conditions

tag: cell line

value: Metastatic melanoma

tag: condition of culture

value: Hypoxia


title: mTGN_ctrl_1_1

value: neurons


title: 574_D

value: PPARD agonist


title: 574_A

value: PPARA agonist


title: SKMEL5NOR2

value: Normoxia


title: SKMEL5HYP1


title: 1068


title: 574_CTR

value: Untreated


title: Pam3CSK4_3

value: trigeminal ganglion

value: Pam3CSK4 (1 microg/ml) treated


title: 1063

value: Blood


title: 1066

value: B-cell acute lymphoblastic leukemia


title: SKMEL5NOR1


title: 1052


title: 567_G


title: Pam3CSK4_1


title: Pam3CSK4_2


title: mTGN_RFSL1_1_1

value: FSL-1 (1 microg/ml)  treated


title: 567_D


title: 567_A

description: lane1-s025-index-CAGGAC-28_567_A_-rd_S25


title: Control_3


title: 567_CTR


title: A375NOR2


title: 1043


title: A375HYP1


title: Control_2


title: 1042


title: 576_G


title: A375NOR1


title: A2058NOR2


title: 1032


title: 1018


title: 576_CTR


title: DRG_H_SE3

source_name: mouse dorsal root ganglion neurons

value: dorsal root ganglion

value: serpinE1(20microg/ml) treated


title: A375HYP2


title: 1048


title: DRG_H_SE2


title: DRG_H_SE1


title: 1015


title: 576_D


title: Control_1

value: untreated


title: DRG_C3


title: A2058HYP1


title: A2058NOR1


title: 1010


title: 581_D

description: lane1-s018-index-ACGTCT-21_581_D_-rd_S18

value: Liver


title: 1011


title: DRG_C2


title: DRG_C1


title: A2058HYP2


title: 1003


title: IR+

city: Nanchang

first: Yi

last: Sang

state: Jiangxi

address: No.128,Xiangshan North Road

institute: Nanchang University

description: IR+

library_source: genomic

data_processing:
The sequencing data was processed and filterd with cutadapt(v1.18) leaving only the sgRNA sequence for further analysis
The sgRNA reads were mapped to the  GeCKOv2 sgRNA library with Bowtie2 (v2.3.5)
The count of each mapped reads was calculated by Model-bassed Analysis of Genome-wide CRISPR/Cas9 Knockout(MAGeCK) algorithm
Genome_build: GeCKOv2 sgRNA library (Pooled Library #1000000048, #1000000049)
Supplementary_files_format_and_content: tab-delimited text files include normalized read counts for each Sample ...

molecule: genomic DNA

source_name: lung cancer cell lines

growth_protocol: 10% FBS in DMEM

extract_protocol:
The genomic DNA was extracted by Tissue DNA Kit(Omega, cat: D3396-01)
Libraries were prepared according to Illumina's instructions

value: lung cancer cell lines

tag: passages

tag: strain


title: 576_A


title: IR-

description: IR-


title: HelaEdU48hTF2

status: Public on Aug 02 2022

city: Chapel Hill

first: Yanyan

last: Yang

state: NC

address: 120 Mason Farm Road

department: Biochemistry & Biophysics

institute: University of North Carolina  at Chapel Hill

data_processing:
adaptor trimming
filtering reads based on length
random sampling
alignment
read filtering based on nucleotide composition
strand separation
gene coverage count
Assembly: hg38
Supplementary files format and content: bigWig files
Library strategy: XR-seq

extract_protocol: The detailed protocol was published on Genome-wide mapping of nucleotide excision repair with XR-seq (Nature Protocols volume 14, pages248–282 (2019), https://www.nature.com/articles/s41596-018-0093-7).

value: Edu

value: Hela

tag: time

tag: antibody

value: anti-TFIIH and anti-BrdU


title: HelaEdU24hTF2


title: HelaEdU12hTF2


title: 581_G

value: PPARG agonist


title: HelaEdU6hTF2


title: HelaEdU12hTF1


title: HelaEdU24hTF1

data_processing:
adaptor trimming
filtering reads based on length
random sampling
alignment
read filtering based on nucleotide composition
strand separation
gene coverage count
Assembly: hg38
Supplementary files format and content: no
Library strategy: XR-seq


title: HelaEdU6hTF1


title: GFP- and anti-MXD4 shRNA-transduced [MXD4KD] Bio rep.1

city: Evry

first: Nicolas

last: FORTUNEL

address: 2 Rue Gaston Cremieux

country: France

institute: CEA

data_processing:
Base Calling: Basecalls performed using CASAVA version 1.4
Read Quality: Quality of raw reads was assessed using FastQC quality control version 0.11.3  http://www.bioinformatics.babraham.ac.uk/projects/fastqc
Mapping: Primary assembly Homo sapiens genome sequence (release GRCh38.p3) and transcriptome annotations (Ensembl release 81) were downloaded from the GENCODE website2 (files GRCh38.primary_assembly.genome.fa  and gencode.v23.primary_assembly.annotation.gtf respectively). Raw read data (fastq files) were mapped to these sequences using STAR
Normalization: For the quality control representations, the effective library sizes were first computed using function estimateSizeFactors from package DESeq24. The raw count values were then transformed using a regularized log (function rlogTransformation from package DESeq2 with parameter blind=TRUE).
Gene filtering: Genes that did not have more than one count per million counts in at least 2 samples were filtered out. Genes were also filtered if their Ensembl description was either empty, “Uncharacterized protein” or an open reading frame.
Assembly: GRCh38
Supplementary files format and content: CSV files include raw counts (raw counts)  and cpm values (normalized counts) for each Sample

source_name: skin, epidermis

growth_protocol: Human skin tissue from adult healthy donors was collected in the context of breast reduction surgery, after informed consent. Epidermal keratinocytes were extracted after enzymatic treatment. Frozen banked samples of human epidermal holoclone keratinocytes were studied as a model of skin keratinocyte precursor cells. Mass cultures of holoclone cells (keratinocyte precursor cells) were performed in a serum-containing medium, in the presence of a feeder-layer of human dermal fibroblasts growth-arrested by γ irradiation (60 Gy). Cultures were performed in plastic devices coated with type I collagen. Medium was renewed 3 times a week, and sub-cultured every week. Numbers of population doublings achieved by cultures were calculated after each passage, as follows: PD = (log N/N0)/log 2, where N0 represents the number of plated cells, and N the number of cells obtained after 1 week of growth.

extract_protocol:
Total RNA from these 6 samples was extracted with RNeasy Mini and Micro kits (Qiagen) followed by quality control using capillary electrophoresis (RNA 6000 Nano chips, Agilent).
Libraries were prepared according to Illumina's instructions with theTruSeq RNA Library Prep Kit v2. Briefly, total RNA (1µg) was purified and fragmented before first strand and second strand cDNA synthesis. Then ends are repaired and 3’ends adenylated before adapters ligation and PCR amplification. Libraries were then validated, normalized, and sequenced on Illumina HiSeq 1000 as short-insert paired-end libraries with read lengths 100 bp.

treatment_protocol: Lentiviral vectors were used for stable gene knock-down in holoclone cells (keratinocyte precursor cells). Transduction was performed on cultured cells at 50 population doublings after initial clonal derivation and at 20 % confluence. Cells were incubated overnight with lentiviral particles in the presence of hexadimethrine bromide. After 3 days, keratinocytes at 80 % confluence were collected and analyzed by flow cytometry. Transduced cells were sorted according to their GFP fluorescence.

value: skin, epidermis

value: holoclone cells, keratinocyte precursor cells

tag: genotype

value: MXD4 knockdown


title: GFP- and anti-MXD4 shRNA-transduced [MXD4KD] Bio rep.2

tag: cell type


title: 581_A

description: lane1-s017-index-CAATGC-20_581_A_-rd_S17

value: PPARA agonist


title: LS1034_POU5F1B-shRNA5_rep3

city: Lausanne

first: Evarist

last: Planet

state: Vaud

address: Route Cantonale

country: Switzerland

institute: EPFL

data_processing:
Reads were aligned to the human genome (hg19) with hisat2. Counts of mRNA reads on genes were computed using featureCounts.
Genome_build: hg19
Supplementary_files_format_and_content: tab files contain counts on genes.

molecule: polyA RNA

source_name: cells were transduced with a vector encoding POU5F1B-targeting shRNA5 under a doxycycline inducible promoter

growth_protocol: LS1034 cells were cultured in RPMI 1640 medium (Gibco) supplemented with 10% FCS.

extract_protocol:
TruSeq stranded RNA sample preparation kit (Illumina).
Total RNA was extracted with Trizol (ThermoFisher) followed by a spin column-based RNA purifiaction kit (Roche) and DNase-I treatment (Sigma).
Truseq Stranded mRNA-seq protocol

treatment_protocol: Transduced LS1034 cells were treated with 1 ug/mL doxycycline 72 hours before harvesting.

tag: source

value: Tissue culture

value: LS1034 human colorectal cancer cell line

tag: knock-out and knock-down

value: shRNA5-mediated POU5F1B knock-down


title: LS1034_POU5F1B-shRNA5_rep1


title: LS1034_POU5F1B-shRNA3_rep3

source_name: cells were transduced with a vector encoding POU5F1B-targeting shRNA3 under a doxycycline inducible promoter

value: shRNA3-mediated POU5F1B knock-down


title: LS1034_POU5F1B-shRNA3_rep2


title: GFP-transduced [MXD4WT] Bio rep.2

value: WT


title: GFP-transduced [MXD4WT] Bio rep.3


title: GFP- and anti-MXD4 shRNA-transduced [MXD4KD] Bio rep.3


title: LS1034_POU5F1B-shRNA5_rep2


title: GFP-transduced [MXD4WT] Bio rep.1


title: 581_CTR

tag: treatment

value: Untreated


title: LS1034_sh-scramble_rep3

source_name: cells were transduced with a vector encoding sh-scamble under a doxycycline inducible promoter

value: shRNA-scramble used as control


title: LS1034_POU5F1B-shRNA3_rep1


title: LS1034_sh-scramble_rep2


title: HT29_POU5F1B_rep2

source_name: cells were transduced with a vector encoding POU5F1B under a doxycycline inducible promoter

growth_protocol: HT29 cells were cultured in McCoy’s 5A medium (Thermo Fisher) supplemented with 10% FCS.

treatment_protocol: Transduced GT29 cells were treated with 1 ug/mL doxycycline 72 hours before harvesting.

value: HT29 human colorectal cancer cell line

value: POU5F1B overexpression


title: HT29_POU5F1B_rep1


title: HT29_POU5F1B_rep3


title: HelaEdU9hTF2

value: Edu


title: HelaEdU9hTF1

value: Hela


title: HT29_OCT4_rep3

source_name: cells were transduced with a vector encoding OCT4 under a doxycycline inducible promoter

value: OCT4 overexpression


title: HT29_OCT4_rep1


title: HT29_OCT4_rep2


title: HT29_GFP_rep3

source_name: cells were transduced with a vector encoding GFP under a doxycycline inducible promoter

value: GFP overexpression


title: HT29_GFP_rep2


title: SW480_POU5F1B_rep3

source_name: cells were transduced with a vector encoding POU5F1B under a dox inducible promoter

growth_protocol: SW480 cells were cultured in L-15 Medium (Leibovitz) supplemented with 10% FCS.

extract_protocol:
Total RNA was extracted with Trizol (ThermoFisher) followed by a spin column-based RNA purifiaction kit (Roche) and DNase-I treatment (Sigma).
Truseq Stranded mRNA-seq protocol

treatment_protocol: SW480 transduced cells were treated with 1 ug/mL doxycycline 72 hours before harvesting.

value: human colorectal cancer cell line

tag: genotype/variation


title: SW480_POU5F1B_rep2


title: SW480_POU5F1B_rep1


title: SW480_GFP_rep3

source_name: cells were transduced with a vector encoding GFP under a dox inducible promoter


title: SW480_GFP_rep1


title: SW480_GFP_rep2


title: Vehicle rep2 [RS_10_10_TAGCTT_L001]

city: Dallas

first: W. Lee

last: Kraus

state: TX

address: 5323 Harry Hines Blvd.

institute: UT Southwestern Medical Center

data_processing:
Paired-end Total RNA-seq reads were aligned to the human reference genome (GRCh37/hg19) including autosomes, X chromosome, and one complete copy of an rDNA repeat (GenBank ID: U13369.1) using the spliced read aligner TopHat version 1.4.0 (Trapnell et al., 2009) (default parameters and ‘max-multihitis=1’,’mate-inner-dist=100’).  The reads were aligned to the rDNA repeat to check rRNA contamination in the library; and (3) The output from TopHat was converted into BED files using SAMtools (v.0.1.18) (Li et al., 2009) and BEDTools (v.2.16.2) (Quinlan et al., 2010).
Mapped reads were sorted and captured in bam files for further downstream analysis of differential gene expression and  “wiggle” files counting reads in non-overlapping 200-bp windows across the genome for presentation as genome browser tracks by using the BEDTools software package  (Quinlan et al, 2010)
Genome_build: hg19
Supplementary_files_format_and_content: BigWig

source_name: MCF7_Vehicle 3h

growth_protocol: MCF-7 cells, kindly provided by Benita Katzenellenbogen (University of Illinois, Urbana-Champaign), were maintained in MEM medium with Hank’s salts (Sigma, M1018) supplemented with 5% HyClone calf serum (GE Healthcare, SH30072). For experiments involving estrogen treatment, the cells were grown for at least 3 days in phenol red-free MEM Eagle medium with Earle's salts (Sigma, M3024) supplemented with 5% charcoal-dextran-treated calf serum and then treated with ethanol (vehicle) or 17beta-estradiol (E2; 100 nM).

extract_protocol: Total RNAs are extracted from MCF7 cells treated with 100 nM E2 or Veh for 3h. RNA were extracted with Qiagen Rneasy plus mini kit followed by the Ribo zero kit (Epicenter) to remove rRNA. Each condition has two replicates. They are sequenced on Hiseq Paired-End 100bp x2. The first number after "RS" in the label is the illumina barcode number. They are all sequenced in one lane.

value: ethanol (vehicle) for 3h


title: Vehicle rep1 [RS_5_5_ACAGTG_L001]


title: E2 3h rep2 [RS_11_11_GGCTAC_L001]

source_name: MCF7_E2 3h

value: 17beta-estradiol (E2; 100 nM) for 3h


title: E2 3h rep1 [RS_6_6_GCCAAT_L001]


title: LS1034_sh-scramble_rep1

tag: tissue


title: Vanc_16

city: New York

first: Julia

last: Brown

state: New York

address: 413 E 69th Street, 1252A

institute: Weill Cornell Medicine

data_processing:
Library strategy: 16S rRNA seq
Paired-end reads were analyzed and classified into operational taxonomic units (OTUs) at > 97% identity level using Mothur v.1.40.5
Taxonomic assignments were determined using the SILVA 16S rRNA reference file release 132 and the Ribosomal Database Project (RDP) training set version 16
Genome_build: SILVA 16S rRNA reference file release 132
Supplementary_files_format_and_content: OTU table xlsx file

source_name: Fecal DNA

extract_protocol:
DNA was extracted from freshly collected fecal pellets using the Omega Bio-Tek E.Z.N.A. stool DNA kit
RNA libraries were prepared for sequencing using standard Illumina 16S Metagenomic Sequencing Library Preparation protocols

value: Vancomycin


title: Vanc_01


title: Vanc_18


title: Vanc_07


title: Gent_04

value: Gentamicin


title: Gent_06


title: Gent_03


title: Gent_21


title: HT29_GFP_rep1

value: Tissue culture


title: Ctrl_20

value: Control


title: Ctrl_19


title: Vanc_14


title: Gent_09


title: AW2, wildtype, ATAC-seq

city: Baltimore

first: Jonathan

middle: Joseph

last: Augustin

state: MD

address: 733 N. Broadway Rm#332

institute: Johns Hopkins Medical Institute

data_processing:
Sequenced libraries were aligned to the mouse genome (UCSC mm10) using the bowtie2 short read aligner. From here, bam files were generated and indexed using samtools, and bigwig files were generated using bamCoverage. Peaks were called using MACS-3 and differential peak analysis was performed in R using DiffBind. Motif enrichment analysis was performed using the R implementation of MEME Suite’s Analysis of Motif Enrichment test.
Assembly: mm10
Supplementary files format and content: bigwig

source_name: cortical neurospheres

extract_protocol:
Neurospheres were passaged using 1X TrypLE Express (Thermofisher cat#:12604013) with gentle trituration and passed through a 40µm cell strainer. For ATAC-seq library preparation, we utilized the Omni-ATAC-seq protocol first described by (Buenrostro et al., 2013, 2015), with minor changes. For each biological replicate, 50,000 cells were counted and washed in ice-cold 1ml 1X PBS. To isolate nuclei, the cells were pelleted and resuspended in 50μl of ice cold Lysis Buffer (10mM Tris-HCl, pH 7.5, 10mM NaCl, 3mM MgCl2, 0.1% NP-40, 0.1% Tween-20, 0.01% Digitonin) and gently mixed by pipet 3 times to resuspend. Cells were incubated on ice in Lysis Buffer for 3 minutes before being washed with 1ml Wash Buffer (10mM Tris-HCl, pH 7.5, 10mM NaCl, 3mM MgCl2, 0.1% Tween-20) 3 times to stop the lysis reaction. Nuclei were spun down at 500G for 10 minutes and the supernatant was discarded. For each sample, a Transposition Reaction Mix was made using the Nextera DNA Library Prep Kit (25μl 2X TD Buffer (Tagment DNA Buffer), 16.5μl 1X PBS, 0.5μl 10% Tween-20, 0.5μl 1% Digitonin, 2.5μl Tn5 Transposase (Tagment DNA Enzyme 1), 5μl nuclease-free H2O. Each nuclear pellet was resuspended in the Transposition Reaction Mix and incubated at 37°C for 30 minutes in a heat block with occasional mixing. DNA was purified using a Qiagen MinElute Reaction Cleanup Kit and eluted in 10μl of 10mM Tris-HCl pH 8.0. The isolated DNA was used as the input into the PCR amplification/ Library Generation step. Each PCR reaction consisted of 10μl of purified transposed DNA, 10μl nuclease-free H2O, 2.5μl of Ad1_noMx forward primer, and 2.5μl of the reverse primer. Samples we PCR amplified using the following settings: 72°C for 5 minutes, then 98°C for 30 seconds followed by 16 cycles of (98°C for 10 seconds, 63°C for 30 seconds, 72°C for 1 minute) then 72°C for 5 minutes and hold at 4°C. The number of PCR cycles to use was determined by using qPCR on 1/5th of the library from each sample. After the first 5 cycles above, the libraries were removed and 5μl of the PCR reaction was subjected to qPCR for 20 cycles. The number of cycles needed to reach a third of the maximum fluorescence for each sample was used as the remaining number of cycles to add, in this case, 11 cycles, for a total of 16 cycles.
Libraries were purified using AMPure XP beads to remove primer dimers using the standard protocol. To remove the small fragments, 1.8X bead volume was added to each library. For removing large fragments >1000bp, 0.5X bead volume was used on the eluate obtained above. Once finished, the purified libraries were quality controlled using Agilent High Sensitivity DNA bioanalysis chip to look for nucleosome periodicity and measured using QuBit 2.0 for DNA concentration, both following manufacturer's instructions. Samples were sequenced using 50bp paired-end reading at roughly 50 million reads per sample.

value: cortical neurospheres

value: Wildtype


title: AX3, Pantr2 knockout, ATAC-seq

value: Pantr2 knockout


title: AW1, Pantr2 knockout, ATAC-seq


title: AW4, wildtype, ATAC-seq


title: KO3

city: Shanghai

first: haohao

last: zhang

state: Shanghai

address: 320 Yueyang Road

institute: Shanghai Institutes for Biological Sciences, University of Chinese Academy of Sciences, Chinese Academy of Sciences

data_processing:
Illumina HiSeq platform was used for basecalling
Sequenced reads were trimmed for adaptor sequence, and filtered low-complexity or low-quality sequences out. Clean data were mapped to mm10 whole genome using bowtie2 (v 2.2.5) with parameters -q --phred64 --sensitive --dpad 0 --gbar 99999999 --mp 1,1 --np 1 --score-min L,0,-0.1 -p 16 -k 200
FPKM were calculated using RSEM(v1.2.8).
Genome_build: GCF_000001635.25_GRCm38.p5
Supplementary_files_format_and_content: A tab-delimited text file including FPKM values was generated for all samples

source_name: intestinal TCRβ+CD8αα+ intraepithelial lymphocytes

extract_protocol:
Intestinal TCRβ+CD8αα+ intraepithelial lymphocytes of Kdm6bfl/fl-CD4Cre and Kdm6bfl/fl mice were sorted by flow cytometry. Total RNA was isolated with Trizol.
Transcriptome libraries were constructed for sequencing using standard Illumina protocols.

value: primary intestinal TCR{beta}+CD8{alpha}{alpha}+ intraepithelial lymphocytes

value: small intestine


title: KO1


title: Con3

value: control


title: Osteoblast-hPTH7-34-3

type: RNA

city: Charleston

first: Jeremy

last: Barth

state: SC

address: 173 Ashley Ave, BSB 627

institute: MUSC

hyb_protocol: Fragmented and Labeled ss-DNA (3.5 ug) was hybridized to Affymetrix Mouse Gene 2.0 ST GeneChips overnight at 45oC. Hybridized GeneChips were washed and stained in an Affymetrix Fluidics Station 450

scan_protocol: Microarrays were scanned using an Affymetrix 7G Scanner

data_processing: CEL files were imported into Affymetrix Expression Console software and data were anayzed by RMA (Gene Level Default RMA sketch) to obtain probe set expression values

label: biotin

source_name: osteoblast, hPTH(7-34), 4 hr

label_protocol: Total RNA (100 ng) converted to fragmented and biotin-labeled ssDNA by WT Plus kit (Affymetrix)

growth_protocol: Dissected strips of calvaria from 7-10 day old C57BL/6J mice were incubated at 37oC with shaking for 20 min in a solution of 0.1% collagenase II (Worthington) and 0.05% trypsin (Invitrogen) in αMEM (Invitrogen). Supernatants were neutralized with 25% heat-inactivated fetal bovine serum (FBS, Invitrogen), and the digestion of calvarial strips was repeated twice. Supernatants from the three digestions were combined and centrifuged, and pellets were reconstituted in growth medium consisting of αMEM containing 10% heat-inactivated FBS and 0.1% Primocin (Invivogen). After 24 hours in culture, Primocin was reduced to 0.05%. Cells were sub-cultured at 90% confluency and at passage 2 differentiated post-confluence (Day 3). Cells were differentiated starting on Day 3 in αMEM containing 10% heat-inactivated FBS and 0.05% Primocin supplemented with 5 mMβ-glycerophosphate (Millipore Sigma) and 50mg/L ascorbic acid (Millipore Sigma). On Day 10 cells were serum deprived in medium containing 0.5% BSA (Millipore Sigma) for 24 hrs before experimental treatment.

extract_protocol: RNA was prepared with the RNeasy Plus Mini Kit (Qiagen).  RNA quality was assessed by Bioanalyzer 2100 (Agilent).

treatment_protocol: Cells were treated with vehicle or hormone for 4 hours in serum-free medium containing 0.5% BSA. Treatments were either vehicle, 100 nM human parathyroid hormone (1-34) (hPTH(1-34), Bachem), 100 nM PTH(1-36) (American Peptide), 1 µM bovine (D-Trp12,Tyr34)-PTH(7-34) amide (bPTH(7-34), Bachem), 100 nM bPTH(3-34) (Bachem), 100 nM hPTH(1-31) (Bachem), or 100 nM hPTH(7-34) (Prospec). Following treatment, cells were washed with PBS prior to RNA isolation

tag: cell

value: Osteoblast


title: Osteoblast-bPTH7-34-3

source_name: osteoblast, bPTH(7-34), 4 hr


title: Osteoblast-bPTH7-34-1


title: Osteoblast-hPTH1-31-2

source_name: osteoblast, hPTH(1-31), 4 hr


title: Osteoblast-hPTH1-34-3

source_name: osteoblast, hPTH(1-34), 4 hr


title: Osteoblast-PTH1-36-2

source_name: osteoblast, PTH(1-36), 4 hr


title: Osteoblast-Vehicle 3

source_name: osteoblast, vehicle 4 hr

value: Vehicle


title: Osteoblast-hPTH1-34-2


title: Osteoblast-bPTH7-34-2


title: Osteoblast-PTH1-36-3


title: Osteoblast-hPTH1-31-3


title: Osteoblast-Vehicle 1


title: Con1


title: Osteoblast-hPTH7-34-1


title: Osteoblast-bPTH3-34-3

source_name: osteoblast, bPTH(3-34), 4 hr


title: Osteoblast-bPTH3-34-2


title: Osteoblast-PTH1-36-1


title: Osteoblast-Vehicle 2


title: TB-IH_Diagnosis_South_Africa_SUN121

city: London

first: Jackie

last: Cliff

address: Keppel Street

department: Immunology and Infection

institute: London School of Hygiene & Tropical Medicine

data_processing:
Alignment STAR version 2.5.1b
Quantification HT-seq count version 0.6.1
Normalisation DESeq2 version 1.12.4
Assembly: Human_g1k_v37
Supplementary files format and content: read count for each ensembl gene id, un-normalised, un-filtered

source_name: Whole Blood

extract_protocol:
Whole blood stored in PAXgene Blood RNA collection tubes and extracted using the PAXgene blood miRNA kit (Qiagen)
Bioscientific NEXTflex-Rapid-Directional mRNA-seq sample preparation (Bioscientific)

tag: patient id

value: TB-IH

tag: timepoint

value: Diagnosis

tag: location


title: Osteoblast-hPTH1-34-1


title: Osteoblast-hPTH7-34-2


title: TB-preDM_Week_2_South_Africa_SUN55

value: TB-preDM


title: TB-IH_Month_2_Indonesia_UNPAD2

value: Indonesia


title: TB-preDM_Diagnosis_South_Africa_SUN137


title: TB-IH_Diagnosis_South_Africa_SUN50


title: TB-preDM_Month_6_South_Africa_SUN55


title: TB-IH_Month_6_Indonesia_UNPAD11


title: TB-IH_Month_6_South_Africa_SUN136


title: TB-IH_Month_6_Indonesia_UNPAD15


title: TB-preDM_Diagnosis_South_Africa_SUN55


title: TB-preDM_Month_6_South_Africa_SUN54


title: TB-IH_Week_2_South_Africa_SUN50


title: TB-IH_Week_2_Indonesia_UNPAD13


title: TB-IH_Month_2_South_Africa_SUN121


title: TB-preDM_Month_2_South_Africa_SUN55


title: TB-IH_Month_2_South_Africa_SUN104


title: TB-preDM_Month_6_South_Africa_SUN140


title: TB-IH_Month_6_Indonesia_UNPAD7


title: TB-IH_Diagnosis_South_Africa_SUN60


title: TB-IH_Month_6_South_Africa_SUN119


title: TB-IH_Month_6_South_Africa_SUN116


title: TB-IH_Diagnosis_South_Africa_SUN116


title: TB-IH_Week_2_South_Africa_SUN60


title: TB-IH_Diagnosis_South_Africa_SUN120


title: TB-preDM_Month_2_South_Africa_SUN54


title: TB-IH_Diagnosis_South_Africa_SUN105


title: TB-IH_Diagnosis_Indonesia_UNPAD15


title: TB-IH_Month_2_Indonesia_UNPAD11


title: TB-IH_Month_2_Indonesia_UNPAD15


title: TB-IH_Month_2_South_Africa_SUN127


title: TB-preDM_Week_2_South_Africa_SUN137


title: TB-IH_Month_6_South_Africa_SUN60


title: TB-IH_Week_2_South_Africa_SUN116


title: TB-IH_Week_2_Indonesia_UNPAD7


title: Osteoblast-bPTH3-34-1


title: TB-IH_Diagnosis_South_Africa_SUN119


title: TB-IH_Month_2_South_Africa_SUN136


title: TB-preDM_Month_2_South_Africa_SUN137


title: TB-IH_Month_6_Indonesia_UNPAD23


title: TB-IH_Week_2_Indonesia_UNPAD17


title: TB-preDM_Diagnosis_South_Africa_SUN133


title: TB-IH_Month_2_South_Africa_SUN120


title: TB-IH_Month_6_South_Africa_SUN50


title: TB-IH_Month_2_Indonesia_UNPAD13


title: TB-IH_Diagnosis_Indonesia_UNPAD7


title: TB-IH_Diagnosis_South_Africa_SUN136


title: TB-IH_Month_2_South_Africa_SUN50


title: TB-IH_Month_6_South_Africa_SUN127


title: TB-IH_Week_2_South_Africa_SUN105


title: TB-IH_Month_2_Indonesia_UNPAD23


title: TB-IH_Week_2_South_Africa_SUN120


title: TB-IH_Month_2_Indonesia_UNPAD7


title: TB-IH_Diagnosis_Indonesia_UNPAD2


title: TB-IH_Month_2_South_Africa_SUN105


title: TB-IH_Diagnosis_Indonesia_UNPAD26


title: TB-preDM_Week_2_South_Africa_SUN133


title: TB-IH_Month_6_Indonesia_UNPAD13


title: TB-IH_Diagnosis_South_Africa_SUN104


title: TB-IH_Week_2_Indonesia_UNPAD23


title: TB-IH_Month_6_Indonesia_UNPAD17


title: TB-IH_Week_2_South_Africa_SUN121


title: TB-IH_Week_2_Indonesia_UNPAD26


title: TB-preDM_Month_6_South_Africa_SUN133


title: TB-IH_Week_2_South_Africa_SUN127


title: TB-IH_Month_2_South_Africa_SUN119


title: TB-IH_Month_6_Indonesia_UNPAD2


title: TB-IH_Week_2_Indonesia_UNPAD2


title: TB-IH_Month_6_Indonesia_UNPAD26


title: TB-IH_Week_2_South_Africa_SUN119


title: TB-preDM_Diagnosis_South_Africa_SUN54


title: TB-IH_Month_6_South_Africa_SUN120


title: TB-preDM_Week_2_South_Africa_SUN54


title: TB-preDM_Week_2_South_Africa_SUN140


title: TB-IH_Diagnosis_Indonesia_UNPAD11


title: TB-IH_Diagnosis_Indonesia_UNPAD13


title: TB-IH_Month_6_South_Africa_SUN105


title: TB-IH_Month_6_South_Africa_SUN104


title: TB-preDM_Month_2_South_Africa_SUN140


title: TB-IH_Diagnosis_Indonesia_UNPAD23


title: TB-IH_Diagnosis_South_Africa_SUN127


title: TB-preDM_Diagnosis_South_Africa_SUN140


title: TB-IH_Month_2_South_Africa_SUN116


title: TB-IH_Week_2_South_Africa_SUN104


title: TB-preDM_Month_6_South_Africa_SUN137


title: TB-IH_Month_6_South_Africa_SUN121


title: TB-preDM_Month_2_South_Africa_SUN133


title: H2O_2

city: Ottawa

first: Matthew

last: Meier

state: Ontario

address: 251 Sir Frederick Banting Driveway

department: Health Canada

country: Canada

institute: Government of Canada

description:
Plate 1,Flavour A
H11_S88_R1_001

data_processing:
Base calling was performed by bcl2fastq v 2.20.422
Quality trimming was performed using fastp v0.20.0
Alignment was performed using STAR v2.7.8a
Gene counts were performed with qCount in the QuasR R package v1.30.0
Normalization was performed with DESeq2 v1.30.1
Genome_build: Biospyder S1500 Gene Set
Supplementary_files_format_and_content: count_table.csv is a genes by samples count matrix of raw counts from the TempO-Seq data alignments.

source_name: Human TK6 Cells

growth_protocol: TK6 cells, a spontaneously transformed human lymphoblastoid cell line, were cultured in suspension in RPMI 1640 supplemented with 10% fetal bovine serum and treated with case study chemicals, as described previously (Li et al., 2015; Li et al., 2017). Concisely, exponentially growing cells were treated at a density of 4-5×105 cells per mL in parallel 96-well plates. Following the 4 hour exposure, cells were washed and collected for RNA extraction for subsequent gene expression analysis using NanoString nCounter® and BioSpyder TempO-Seq® gene expression analysis.

extract_protocol:
RNA extraction was performed using the protocol accompanying the TRIzol™ reagent (Invitrogen, part of ThermoFisher Scientific, Waltham, MA USA), which was then purified via an RNA cleanup step using an RNeasy column according to the manufacturer’s instructions (Qiagen, Germantown, MD USA). Purified RNA was quantified and analyzed for quality using a NanoDrop™ spectrophotometer and an Agilent BioAnalyzer, respectively.
TempO-Seq® gene expression analysis was conducted on all non-cytotoxic concentrations of exposed and control TK6 cell lysates. As such, the 500 µM and 1,000 µM concentrations of MTZ and NIT were omitted, in addition to the 1,000 µM concentration of NOV, as these concentrations surpassed the cytotoxicity threshold of 50% cell death. Libraries were prepared in a 96-well plate format using the TempO-Seq® Human Tox+Surrogate Panel reagent kit (BioSpyder Technologies, Carlsbad, CA, USA), following the company’s protocol. Human Brain Total RNA and qPCR Human Reference Total RNA (Takara Bio, CA, USA) were included as positive assay controls and a no-lysate control (1X TempO-Seq® R Lysis Buffer only) was included as a negative assay control (two replicates per control). In order to fit the assay controls on the plate, the lowest concentration (i.e., 2 µM) of each test chemical was eliminated from the analysis. The same positive and negative controls were included for TempO-Seq® analysis as for nCounter® analysis (i.e., 2 mM CA as a negative control; 10 µM BL and 4 Gy IR as positive controls).  In brief, 2 µl of exposed and control cell lysates were hybridized with the targeted Human S1500+Tox Panel detector oligo (DO) probe mix (v1.1; 2,977 probes) in 1X TempO-Seq® R Lysis Buffer for 10 min at 70oC followed by a temperature gradient with a ramp rate of 0.5oC/min to 45oC over a 50 min period, followed by a nuclease digestion at 37oC for 90 min to remove excess, unbound, or incorrectly bound DOs enzymatically. Ligation of the DO pairs bound to adjacent target sequences was then completed with a 60 min incubation at 37oC, immediately followed with a 15 min enzyme denaturation at 80oC to generate a pool of amplification templates. All amplification templates (i.e., 10 µl of ligated DOs) were transferred to its corresponding well of the 96-well PCR Pre-Mix and Primers plate. Amplification proceeded with a CFX96 Real-Time PCR Detection System (Bio-Rad, Mississauga, ON, Canada) to attach a unique sequence tag and the sequencing adaptors to each sample using the following PCR program settings: 37oC for 10 min, 95oC for 2 min; 6 cycles of 95oC for 30 sec, 54oC for 30 sec, 72oC for 120 sec; 16 cycles of 95oC for 30 sec; 72oC for 2 min; 72oC for 1 min. The TempO-Seq® libraries were pooled (5 µl of all 96 samples) and purified using the Macherey-Nagel NucleoSpin® Gel and PCR Clean-Up kit (Clontech Laboratories Inc., Bethlehem, PA, USA), according to the manufacturer’s instructions for PCR cleanup with three modifications outlined in the TempO-Seq® Assay User Guide. The pooled, purified TempO-Seq® libraries were sequenced using a NextSeq® 500/550 High Output flow cell (v2 kit, 75 cycles) on an Illumina NextSeq® 500 Sequencing platform (Illumina, San Diego, CA, USA).
Human TempO-Seq® Tox+ Surrogate (S1500+) - Index Sets A (plate 1); sequenced using an Illumina NextSeq500.

treatment_protocol: Human TK6 cells (ATCC, Manassas, Virginia, USA) were exposed to ten concentrations of each test chemical in triplicate, as follows: 2.0, 3.9, 7.8, 15.6, 31.3, 62.5, 125, 250, 500, and 1,000 µM, in addition to matched vehicle controls. Cells were exposed to the extended concentration range of each chemical for 4 hr in parallel 96-well plates  for evaluation of DNA damage by CometChip® and for the collection of cells for gene expression analysis using nCounter® and TempO-Seq® for TGx-DDI classification. Concisely, exponentially growing cells were treated at a density of 4-5×105 cells per mL in parallel 96-well plates. Following the 4 hr exposure, cells were washed and collected for RNA extraction for subsequent gene expression analysis using NanoString nCounter® and BioSpyder TempO-Seq® gene expression analysis.

tag: cell line

value: Human TK6 Cells

value: Novobiocin (NOV)

tag: concentration

value: 0 µM

tag: index

value: TCAGCTAAT

value: ATGCTCACA


title: NIT_125uM_3

description:
Plate 1,Flavour A
B12_S90_R1_001

value: Nitrofurantoin (NIT)

value: GTAGCTCCA

value: TAACGCGTC


title: NOV_3.9uM_3

description:
Plate 1,Flavour A
C12_S91_R1_001

value: TCTAACCAT


title: HOAC_1600X_3

description:
Plate 1,Flavour A
E12_S93_R1_001

value: Metronidazole (MTZ)

value: GATTGTGTT


title: NOV_62.5uM_3

description:
Plate 1,Flavour A
D12_S92_R1_001

value: GCCGTTACA


title: MTZ_250uM_2

description:
Plate 1,Flavour A
G11_S87_R1_001

value: AGACACAAT


title: NIT_7.8uM_3

description:
Plate 1,Flavour A
A12_S89_R1_001

value: ACGACTTGG


title: MTZ_250uM_3

description:
Plate 1,Flavour A
G12_S95_R1_001


title: TB-IH_Month_2_South_Africa_SUN60

value: TB-IH


title: TB-IH_Diagnosis_Indonesia_UNPAD17

value: Diagnosis


title: NOV_62.5uM_2

description:
Plate 1,Flavour A
D11_S84_R1_001


title: MTZ_15.6uM_2

description:
Plate 1,Flavour A
F11_S86_R1_001

value: CGTAACTTA


title: NIT_7.8uM_2

description:
Plate 1,Flavour A
A11_S81_R1_001

value: Human TK6 Cells


title: NIT_125uM_2

description:
Plate 1,Flavour A
B11_S82_R1_001


title: MTZ_250uM_1

description:
Plate 1,Flavour A
G10_S79_R1_001

value: TGCTTATTC


title: HOAC_1600X_1

description:
Plate 1,Flavour A
E10_S77_R1_001

value: 0 µM


title: MTZ_15.6uM_1

description:
Plate 1,Flavour A
F10_S78_R1_001


title: NOV_62.5uM_1

description:
Plate 1,Flavour A
D10_S76_R1_001

value: Novobiocin (NOV)


title: NIT_125uM_1

description:
Plate 1,Flavour A
B10_S74_R1_001


title: NOV_3.9uM_2

description:
Plate 1,Flavour A
C11_S83_R1_001

value: TCAGCTAAT


title: HOAC_1600X_2

description:
Plate 1,Flavour A
E11_S85_R1_001


title: H2O_1

description:
Plate 1,Flavour A
H10_S80_R1_001

value: ATGCTCACA


title: NOV_3.9uM_1

description:
Plate 1,Flavour A
C10_S75_R1_001


title: NIT_7.8uM_1

description:
Plate 1,Flavour A
A10_S73_R1_001

value: Nitrofurantoin (NIT)


title: MTZ_15.6uM_3

description:
Plate 1,Flavour A
F12_S94_R1_001

value: GTAGCTCCA


title: H2O_3

description:
Plate 1,Flavour A
H12_S96_R1_001


title: Caffeine_2mM_3

description:
Plate 1,Flavour A
H09_S72_R1_001

value: Caffeine (CA; Negative Control)

value: GCGATAGTG


title: MTZ_125uM_3

description:
Plate 1,Flavour A
G09_S71_R1_001

value: Metronidazole (MTZ)


title: NIT_62.5uM_3

description:
Plate 1,Flavour A
B09_S66_R1_001

value: TAACGCGTC


title: MTZ_7.8uM_3

description:
Plate 1,Flavour A
F09_S70_R1_001


title: NOV_31.3uM_2

description:
Plate 1,Flavour A
D08_S60_R1_001

value: TTCCGCACC

value: GCCGTTACA


title: Caffeine_2mM_2

description:
Plate 1,Flavour A
H08_S64_R1_001


title: NOV_500uM_3

description:
Plate 1,Flavour A
E09_S69_R1_001

value: GATTGTGTT


title: MTZ_125uM_2

description:
Plate 1,Flavour A
G08_S63_R1_001

value: AGACACAAT


title: (+) UHRR_2

description:
Plate 1,Flavour A
C09_S67_R1_001

value: No cells; Universal human +ve control

value: No cells; Universal human +ve control_0

value: TCTAACCAT


title: NOV_500uM_2

description:
Plate 1,Flavour A
E08_S61_R1_001

value: Human TK6 Cells


title: NOV_31.3uM_3

description:
Plate 1,Flavour A
D09_S68_R1_001

value: Novobiocin (NOV)


title: MTZ_7.8uM_1

description:
Plate 1,Flavour A
F07_S54_R1_001

value: ATCGCGACT

value: CGTAACTTA


title: NIT_62.5uM_2

description:
Plate 1,Flavour A
B08_S58_R1_001

value: Nitrofurantoin (NIT)


title: NIT_3.9uM_2

description:
Plate 1,Flavour A
A08_S57_R1_001

value: ACGACTTGG


title: NIT_62.5uM_1

description:
Plate 1,Flavour A
B07_S50_R1_001


title: NOV_31.3uM_1

description:
Plate 1,Flavour A
D07_S52_R1_001


title: NOV_500uM_1

description:
Plate 1,Flavour A
E07_S53_R1_001


title: MTZ_125uM_1

description:
Plate 1,Flavour A
G07_S55_R1_001


title: NOV_2uM_1

description:
Plate 1,Flavour A
C07_S51_R1_001

value: 2 µM


title: NOV_250uM_3

description:
Plate 1,Flavour A
E06_S45_R1_001

value: TAGCTCTTC


title: MTZ_62.5uM_3

description:
Plate 1,Flavour A
G06_S47_R1_001

value: Metronidazole (MTZ)


title: NIT_3.9uM_3

description:
Plate 1,Flavour A
A09_S65_R1_001

value: GCGATAGTG


title: Caffeine_2mM_1

description:
Plate 1,Flavour A
H07_S56_R1_001

value: Caffeine (CA; Negative Control)

value: ATGCTCACA


title: Bleo_10uM_3

description:
Plate 1,Flavour A
H06_S48_R1_001

value: Bleomycin (BL, Positive Control)

value: 10 µM


title: MTZ_3.9uM_3

description:
Plate 1,Flavour A
F06_S46_R1_001


title: NIT_3.9uM_1

description:
Plate 1,Flavour A
A07_S49_R1_001


title: MTZ_7.8uM_2

description:
Plate 1,Flavour A
F08_S62_R1_001

value: TTCCGCACC


title: H2O_800X_3

description:
Plate 1,Flavour A
C06_S43_R1_001

value: 0 µM


title: (+) UHRR_1

description:
Plate 1,Flavour A
C08_S59_R1_001

value: No cells; Universal human +ve control

value: No cells; Universal human +ve control_0

value: TCTAACCAT


title: NIT_31.3uM_3

description:
Plate 1,Flavour A
B06_S42_R1_001

value: Human TK6 Cells

value: TAACGCGTC


title: Bleo_10uM_2

description:
Plate 1,Flavour A
H05_S40_R1_001

value: TCCTTCGTG


title: (-) Lysis_2

description:
Plate 1,Flavour A
A06_S41_R1_001

value: No cells; Negative control

value: No cells; Negative control_0

value: ACGACTTGG


title: NIT_31.3uM_2

description:
Plate 1,Flavour A
B05_S34_R1_001

value: Nitrofurantoin (NIT)


title: MTZ_62.5uM_2

description:
Plate 1,Flavour A
G05_S39_R1_001

value: Metronidazole (MTZ)

value: AGACACAAT


title: NOV_15.6uM_2

description:
Plate 1,Flavour A
D05_S36_R1_001

value: Novobiocin (NOV)

value: GCCGTTACA


title: MTZ_3.9uM_2

description:
Plate 1,Flavour A
F05_S38_R1_001

value: CGTAACTTA


title: NOV_250uM_2

description:
Plate 1,Flavour A
E05_S37_R1_001

value: GATTGTGTT


title: Osteoblast-hPTH1-31-1

value: Osteoblast


title: NOV_15.6uM_3

description:
Plate 1,Flavour A
D06_S44_R1_001

value: TAGCTCTTC


title: Bleo_10uM_1

description:
Plate 1,Flavour A
H04_S32_R1_001

value: Bleomycin (BL, Positive Control)

value: 10 µM

value: GCATGGACC

value: ATGCTCACA


title: MTZ_62.5uM_1

description:
Plate 1,Flavour A
G04_S31_R1_001

value: Human TK6 Cells


title: NOV_7.8uM_2

description:
Plate 1,Flavour A
D02_S12_R1_001

value: GTGATCTGA


title: NOV_125uM_2

description:
Plate 1,Flavour A
E02_S13_R1_001


title: DMSO_800X_3

description:
Plate 1,Flavour A
A03_S17_R1_001

value: 0 µM

value: GGAATGCAT


title: MTZ_31.3uM_2

description:
Plate 1,Flavour A
G02_S15_R1_001


title: NOV_15.6uM_1

description:
Plate 1,Flavour A
D04_S28_R1_001


title: NIT_2uM_1

description:
Plate 1,Flavour A
A04_S25_R1_001

value: Nitrofurantoin (NIT)

value: 2 µM

value: ACGACTTGG


title: NOV_125uM_3

description:
Plate 1,Flavour A
E03_S21_R1_001

value: Novobiocin (NOV)


title: H2O_800X_1

description:
Plate 1,Flavour A
C04_S27_R1_001

value: TCTAACCAT


title: NIT_250uM_3

description:
Plate 1,Flavour A
C03_S19_R1_001


title: IR_2

description:
Plate 1,Flavour A
H02_S16_R1_001

value: Ionizing Radiation (IR; positive control)

value: 4 Gy


title: IR_3

description:
Plate 1,Flavour A
H03_S24_R1_001


title: (+) Brain_1

description:
Plate 1,Flavour A
F02_S14_R1_001

value: No cells; Brain +ve control

value: No cells; Brain +ve control_0

value: CGTAACTTA


title: (+) Brain_2

description:
Plate 1,Flavour A
F03_S22_R1_001


title: NIT_15.6uM_3

description:
Plate 1,Flavour A
B03_S18_R1_001

value: TAACGCGTC


title: NOV_250uM_1

description:
Plate 1,Flavour A
E04_S29_R1_001

value: GCATGGACC

value: GATTGTGTT


title: MTZ_31.3uM_3

description:
Plate 1,Flavour A
G03_S23_R1_001

value: Human TK6 Cells

value: Metronidazole (MTZ)

value: AGACACAAT


title: NIT_31.3uM_1

description:
Plate 1,Flavour A
B04_S26_R1_001


title: MTZ_3.9uM_1

description:
Plate 1,Flavour A
F04_S30_R1_001


title: NIT_15.6uM_2

description:
Plate 1,Flavour A
B02_S10_R1_001

value: GTGATCTGA


title: NIT_250uM_2

description:
Plate 1,Flavour A
C02_S11_R1_001

value: Nitrofurantoin (NIT)


title: IR_1

description:
Plate 1,Flavour A
H01_S8_R1_001

value: GCTGTAGTT

value: ATGCTCACA


title: DMSO_800X_2

description:
Plate 1,Flavour A
A02_S9_R1_001

value: 0 µM

value: ACGACTTGG


title: MTZ_31.3uM_1

description:
Plate 1,Flavour A
G01_S7_R1_001


title: (-) Lysis_1

description:
Plate 1,Flavour A
A05_S33_R1_001

value: No cells; Negative control

value: No cells; Negative control_0

value: TCCTTCGTG


title: H2O_800X_2

description:
Plate 1,Flavour A
C05_S35_R1_001

value: Novobiocin (NOV)

value: TCTAACCAT


title: NOV_7.8uM_3

description:
Plate 1,Flavour A
D03_S20_R1_001

value: GGAATGCAT

value: GCCGTTACA


title: MTZ_2uM_1

description:
Plate 1,Flavour A
F01_S6_R1_001

value: 2 µM

value: CGTAACTTA


title: NOV_125uM_1

description:
Plate 1,Flavour A
E01_S5_R1_001

value: Human TK6 Cells

value: GATTGTGTT


title: NOV_7.8uM_1

description:
Plate 1,Flavour A
D01_S4_R1_001


title: NIT_15.6uM_1

description:
Plate 1,Flavour A
B01_S2_R1_001

value: TAACGCGTC


title: TB-only_Diagnosis_Indonesia_UNPAD20

data_processing:
Alignment STAR version 2.5.1b
Quantification HT-seq count version 0.6.1 f=bam, r=name, s=rev, m=union, i=gene id
Normalisation DESeq2 version 1.12.4
Genome_build: Human_g1k_v37
Supplementary_files_format_and_content: read count for each ensembl gene id, un-normalised, un-filtered

value: TB-only

value: Diagnosis

value: Indonesia


title: TB-DM_Diagnosis_Indonesia_UNPAD82

value: TB-DM


title: TB-only_Diagnosis_Indonesia_UNPAD3


title: TB-only_Diagnosis_Indonesia_UNPAD34


title: TB-DM_Week_2_South Africa_SUN112

value: South Africa


title: TB-DM_Diagnosis_South Africa_SUN144


title: TB-only_Month_2_Indonesia_UNPAD30


title: TB-DM_Week_2_Indonesia_UNPAD4


title: TB-DM_Diagnosis_Indonesia_UNPAD19


title: TB-only_Diagnosis_Indonesia_UNPAD30


title: TB-DM_Month_6_Indonesia_UNPAD29


title: TB-DM_Month_2_Indonesia_UNPAD82


title: TB-DM_Diagnosis_South Africa_SUN68


title: TB-only_Month_6_Indonesia_UNPAD14


title: TB-only_Month_2_Indonesia_UNPAD3


title: TB-DM_Week_2_Indonesia_UNPAD29


title: DMSO_800X_1

description:
Plate 1,Flavour A
A01_S1_R1_001

value: Nitrofurantoin (NIT)

value: 0 µM

value: GCTGTAGTT

value: ACGACTTGG


title: TB-DM_Month_2_South Africa_SUN162


title: TB-DM_Month_6_Indonesia_UNPAD6


title: TB-DM_Diagnosis_Indonesia_UNPAD25


title: TB-DM_Week_2_South Africa_SUN144


title: TB-DM_Week_2_South Africa_SUN163


title: TB-DM_Month_6_South Africa_SUN126


title: TB-DM_Month_2_Indonesia_UNPAD8


title: NIT_250uM_1

description:
Plate 1,Flavour A
C01_S3_R1_001

value: TCTAACCAT


title: TB-DM_Month_6_Indonesia_UNPAD24


title: TB-only_Month_6_South Africa_SUN103


title: TB-DM_Month_2_Indonesia_UNPAD10


title: TB-DM_Diagnosis_Indonesia_UNPAD12


title: TB-DM_Month_2_South Africa_SUN59


title: TB-only_Month_6_Indonesia_UNPAD27


title: TB-DM_Diagnosis_Indonesia_UNPAD32


title: TB-DM_Month_2_Indonesia_UNPAD6


title: TB-DM_Diagnosis_Indonesia_UNPAD4


title: TB-DM_Diagnosis_Indonesia_UNPAD10


title: TB-DM_Week_2_Indonesia_UNPAD24


title: TB-only_Week_2_South Africa_SUN103


title: TB-only_Diagnosis_South Africa_SUN103


title: TB-DM_Month_2_South Africa_SUN135


title: TB-DM_Month_6_Indonesia_UNPAD4


title: TB-only_Diagnosis_Indonesia_UNPAD27


title: TB-only_Month_6_South Africa_SUN141


title: TB-DM_Month_2_Indonesia_UNPAD4


title: TB-DM_Diagnosis_South Africa_SUN162


title: TB-only_Diagnosis_South Africa_SUN141


title: TB-only_Week_2_South Africa_SUN132


title: TB-DM_Month_6_South Africa_SUN144


title: TB-DM_Month_2_Indonesia_UNPAD18


title: TB-DM_Diagnosis_Indonesia_UNPAD28


title: TB-DM_Week_2_South Africa_SUN159


title: TB-DM_Month_6_South Africa_SUN149


title: TB-DM_Diagnosis_Indonesia_UNPAD18


title: TB-DM_Diagnosis_South Africa_SUN51


title: TB-only_Week_2_Indonesia_UNPAD30


title: TB-DM_Diagnosis_South Africa_SUN71


title: TB-DM_Month_6_Indonesia_UNPAD31


title: TB-DM_Month_2_South Africa_SUN68


title: TB-only_Month_2_South Africa_SUN103


title: TB-DM_Month_2_South Africa_SUN112


title: TB-DM_Week_2_South Africa_SUN162


title: TB-DM_Month_6_South Africa_SUN51


title: TB-only_Month_2_South Africa_SUN141


title: TB-DM_Month_6_South Africa_SUN163


title: TB-DM_Month_2_Indonesia_UNPAD24


title: TB-DM_Month_6_Indonesia_UNPAD19


title: TB-DM_Month_2_South Africa_SUN51


title: TB-DM_Week_2_South Africa_SUN155


title: TB-DM_Month_2_Indonesia_UNPAD28


title: TB-DM_Diagnosis_Indonesia_UNPAD83


title: TB-DM_Month_2_South Africa_SUN159


title: TB-DM_Diagnosis_Indonesia_UNPAD84


title: TB-only_Week_2_Indonesia_UNPAD20


title: TB-DM_Month_2_South Africa_SUN163


title: TB-DM_Month_2_Indonesia_UNPAD84


title: TB-DM_Diagnosis_Indonesia_UNPAD24


title: TB-DM_Diagnosis_Indonesia_UNPAD6


title: TB-only_Month_2_Indonesia_UNPAD27


title: TB-DM_Month_6_South Africa_SUN135


title: TB-DM_Week_2_South Africa_SUN135


title: TB-DM_Week_2_Indonesia_UNPAD32


title: TB-DM_Week_2_Indonesia_UNPAD82


title: TB-DM_Month_2_Indonesia_UNPAD29


title: TB-DM_Month_6_Indonesia_UNPAD83


title: TB-only_Month_2_South Africa_SUN129


title: TB-only_Diagnosis_South Africa_SUN132


title: TB-DM_Diagnosis_Indonesia_UNPAD8


title: TB-DM_Month_2_South Africa_SUN155


title: TB-DM_Week_2_Indonesia_UNPAD28


title: TB-DM_Month_6_South Africa_SUN68


title: TB-DM_Diagnosis_South Africa_SUN126


title: TB-only_Week_2_South Africa_SUN141


title: TB-DM_Diagnosis_South Africa_SUN135


title: TB-DM_Month_6_South Africa_SUN71


title: TB-DM_Month_2_Indonesia_UNPAD32


title: TB-only_Week_2_Indonesia_UNPAD16


title: TB-DM_Month_2_Indonesia_UNPAD83


title: TB-only_Month_2_Indonesia_UNPAD34


title: TB-DM_Week_2_South Africa_SUN71


title: TB-DM_Week_2_Indonesia_UNPAD10


title: TB-DM_Week_2_Indonesia_UNPAD6


title: TB-DM_Month_6_Indonesia_UNPAD18


title: TB-only_Month_6_Indonesia_UNPAD34


title: EHUVEC_Mock_72h_3_73

city: Richland

first: Natalie

last: Heller

address: 902 Battelle Blvd.

institute: PNNL

hyb_protocol: Hybridization was performed according to Agilent One-Color Microarray-Based Gene Expression Analysis Protocol (Agilent Technology). The labeled cRNAs were purified by RNeasy Mini Kit (Qiagen). The concentration and specific activity of the labeled cRNAs (pmol Cy3/μg cRNA) were measured by NanoDrop ND-1000. 1 μg of each labeled cRNA was fragmented by adding 11 μl 10 × Blocking Agent and 2.2 μl of 25×Fragmentation Buffer, then heated at 60 °C for 30 min, and finally 55 μl 2 × GE Hybridization buffer was added to dilute the labeled cRNA. 100μl of hybridization solution was dispensed into the gasket slide and assembled to the gene expression microarray slide. The slides were incubated for 17 hours at 65°C in an Agilent Hybridization Oven.

scan_protocol: The hybridized arrays were washed, fixed and scanned with using the Agilent DNA Microarray Scanner (part number G2505C).

data_processing: Scanned images were analyzed using Agilent Feature Extraction Software (v11.0.1.1). The limma package for R (available on Bioconductor) was used to perform background correction, quantile normalization (normalizeBetweenArrays), and summarization (avereps) to derive a single normalized intensity value per probe. Outlier samples were detected using PCA and by visual inspection of heatmaps, and all data was re-processed after removing outlier samples. All data processing for each of the biological replicates was performed independently of the other.

source_name: HUVEC cells, mock-inoculated, 72h, bioreplicate 3

label_protocol: Total RNA was labeled according to Agilent’s Quick Amp Labeling protocol as part of the Agilent One-Color Microarray-Based Gene Expression Analysis Protocol (Agilent Technology). Total RNA from each sample was linearly amplified and labeled with Cy3-UTP.

growth_protocol: Cells and virus were grown on 10% FBS DMEM supplemented with L-glut, Pen/Strep, nonessential amino acids and sodium pyruvate.

extract_protocol: RNA was extracted with 1mL of Trizol.

treatment_protocol: Cells were infected with a multiplicity of infection of 10.

tag: virus

value: Mock


title: EHUVEC_Mock_72h_2_72

source_name: HUVEC cells, mock-inoculated, 72h, bioreplicate 2

tag: time


title: EHUVEC_Mock_72h_4_74

source_name: HUVEC cells, mock-inoculated, 72h, bioreplicate 4


title: TB-DM_Month_6_Indonesia_UNPAD10


title: EHUVEC_Mock_48h_4_69

source_name: HUVEC cells, mock-inoculated, 48h, bioreplicate 4


title: EHUVEC_Mock_72h_5_75

source_name: HUVEC cells, mock-inoculated, 72h, bioreplicate 5


title: TB-only_Week_2_Indonesia_UNPAD14


title: EHUVEC_Mock_72h_1_71

source_name: HUVEC cells, mock-inoculated, 72h, bioreplicate 1


title: EHUVEC_Mock_48h_3_68

source_name: HUVEC cells, mock-inoculated, 48h, bioreplicate 3


title: EHUVEC_Mock_48h_1_66

source_name: HUVEC cells, mock-inoculated, 48h, bioreplicate 1


title: EHUVEC_Mock_24h_4_64

source_name: HUVEC cells, mock-inoculated, 24h, bioreplicate 4


title: TB-DM_Month_6_Indonesia_UNPAD84


title: EHUVEC_Mock_24h_3_63

source_name: HUVEC cells, mock-inoculated, 24h, bioreplicate 3


title: EHUVEC_Mock_48h_2_67

source_name: HUVEC cells, mock-inoculated, 48h, bioreplicate 2


title: EHUVEC_Mock_24h_2_62

source_name: HUVEC cells, mock-inoculated, 24h, bioreplicate 2


title: EHUVEC_Mock_48h_5_70

source_name: HUVEC cells, mock-inoculated, 48h, bioreplicate 5


title: EHUVEC_Mock_24h_1_61

source_name: HUVEC cells, mock-inoculated, 24h, bioreplicate 1


title: EHUVEC_Mock_24h_5_65

source_name: HUVEC cells, mock-inoculated, 24h, bioreplicate 5


title: TB-DM_Month_6_South Africa_SUN112

source_name: Whole Blood


title: TB-DM_Diagnosis_Indonesia_UNPAD31


title: TB-DM_Month_2_South Africa_SUN144


title: TB-DM_Week_2_South Africa_SUN68


title: EHUVEC_Mock_8h_5_60

source_name: HUVEC cells, mock-inoculated, 8h, bioreplicate 5


title: TB-DM_Week_2_South Africa_SUN149


title: EHUVEC_Mock_8h_3_58

source_name: HUVEC cells, mock-inoculated, 8h, bioreplicate 3


title: EHUVEC_Mock_8h_4_59

source_name: HUVEC cells, mock-inoculated, 8h, bioreplicate 4


title: TB-DM_Diagnosis_South Africa_SUN159


title: TB-only_Month_2_Indonesia_UNPAD20


title: TB-only_Month_6_Indonesia_UNPAD20


title: TB-DM_Month_2_South Africa_SUN71


title: EHUVEC_Mock_8h_2_57

source_name: HUVEC cells, mock-inoculated, 8h, bioreplicate 2


title: EHUVEC_Mock_8h_1_56

source_name: HUVEC cells, mock-inoculated, 8h, bioreplicate 1


title: TB-DM_Diagnosis_South Africa_SUN149


title: TB-DM_Month_2_South Africa_SUN149


title: TB-DM_Week_2_Indonesia_UNPAD31


title: TB-DM_Month_6_South Africa_SUN59


title: TB-DM_Month_2_Indonesia_UNPAD12


title: TB-DM_Month_6_Indonesia_UNPAD28


title: EHUVEC_Mock_0h_5_55

source_name: HUVEC cells, mock-inoculated, 0h, bioreplicate 5


title: TB-DM_Week_2_Indonesia_UNPAD18


title: TB-only_Week_2_Indonesia_UNPAD3


title: TB-DM_Month_6_Indonesia_UNPAD25


title: EHUVEC_Mock_0h_2_52

source_name: HUVEC cells, mock-inoculated, 0h, bioreplicate 2


title: EHUVEC_deltaVP30WT_72h_5_50

source_name: HUVEC cells, deltaVP30-WT-inoculated, 72h, bioreplicate 5


title: EHUVEC_deltaVP30WT_72h_3_48

source_name: HUVEC cells, deltaVP30-WT-inoculated, 72h, bioreplicate 3


title: TB-DM_Month_2_Indonesia_UNPAD25


title: EHUVEC_Mock_0h_1_51

source_name: HUVEC cells, mock-inoculated, 0h, bioreplicate 1


title: EHUVEC_Mock_0h_4_54

source_name: HUVEC cells, mock-inoculated, 0h, bioreplicate 4


title: EHUVEC_Mock_0h_3_53

source_name: HUVEC cells, mock-inoculated, 0h, bioreplicate 3


title: EHUVEC_deltaVP30WT_72h_4_49

source_name: HUVEC cells, deltaVP30-WT-inoculated, 72h, bioreplicate 4


title: EHUVEC_deltaVP30WT_72h_2_47

source_name: HUVEC cells, deltaVP30-WT-inoculated, 72h, bioreplicate 2


title: EHUVEC_deltaVP30WT_48h_5_45

source_name: HUVEC cells, deltaVP30-WT-inoculated, 48h, bioreplicate 5


title: TB-only_Diagnosis_South Africa_SUN129


title: TB-DM_Month_2_Indonesia_UNPAD19


title: EHUVEC_deltaVP30WT_48h_3_43

source_name: HUVEC cells, deltaVP30-WT-inoculated, 48h, bioreplicate 3


title: TB-DM_Week_2_Indonesia_UNPAD25

source_name: Whole Blood


title: EHUVEC_deltaVP30WT_48h_4_44

source_name: HUVEC cells, deltaVP30-WT-inoculated, 48h, bioreplicate 4


title: TB-only_Month_2_Indonesia_UNPAD16


title: TB-DM_Week_2_South Africa_SUN51


title: EHUVEC_deltaVP30WT_48h_2_42

source_name: HUVEC cells, deltaVP30-WT-inoculated, 48h, bioreplicate 2


title: TB-only_Month_6_South Africa_SUN132


title: TB-DM_Diagnosis_South Africa_SUN112


title: TB-DM_Month_2_Indonesia_UNPAD31


title: EHUVEC_deltaVP30WT_72h_1_46

source_name: HUVEC cells, deltaVP30-WT-inoculated, 72h, bioreplicate 1


title: TB-only_Diagnosis_Indonesia_UNPAD14


title: EHUVEC_deltaVP30WT_24h_3_38

source_name: HUVEC cells, deltaVP30-WT-inoculated, 24h, bioreplicate 3


title: TB-only_Month_2_South Africa_SUN132


title: TB-DM_Month_6_Indonesia_UNPAD8


title: EHUVEC_deltaVP30WT_24h_5_40

source_name: HUVEC cells, deltaVP30-WT-inoculated, 24h, bioreplicate 5


title: EHUVEC_deltaVP30WT_24h_1_36

source_name: HUVEC cells, deltaVP30-WT-inoculated, 24h, bioreplicate 1


title: EHUVEC_deltaVP30WT_24h_4_39

source_name: HUVEC cells, deltaVP30-WT-inoculated, 24h, bioreplicate 4


title: TB-DM_Diagnosis_South Africa_SUN59


title: EHUVEC_deltaVP30WT_8h_4_34

source_name: HUVEC cells, deltaVP30-WT-inoculated, 8h, bioreplicate 4


title: TB-DM_Diagnosis_Indonesia_UNPAD29


title: TB-DM_Week_2_South Africa_SUN59


title: EHUVEC_deltaVP30WT_8h_5_35

source_name: HUVEC cells, deltaVP30-WT-inoculated, 8h, bioreplicate 5


title: TB-only_Month_6_Indonesia_UNPAD30


title: EHUVEC_deltaVP30WT_8h_1_31

source_name: HUVEC cells, deltaVP30-WT-inoculated, 8h, bioreplicate 1


title: TB-DM_Month_6_Indonesia_UNPAD82


title: EHUVEC_deltaVP30WT_0h_5_30

source_name: HUVEC cells, deltaVP30-WT-inoculated, 0h, bioreplicate 5


title: TB-DM_Week_2_Indonesia_UNPAD12


title: EHUVEC_deltaVP30WT_8h_2_32

source_name: HUVEC cells, deltaVP30-WT-inoculated, 8h, bioreplicate 2


title: EHUVEC_deltaVP30WT_0h_3_28

source_name: HUVEC cells, deltaVP30-WT-inoculated, 0h, bioreplicate 3


title: TB-DM_Month_2_South Africa_SUN126


title: TB-only_Week_2_Indonesia_UNPAD34


title: EHUVEC_deltaVP30WT_0h_2_27

source_name: HUVEC cells, deltaVP30-WT-inoculated, 0h, bioreplicate 2


title: TB-only_Month_6_Indonesia_UNPAD16


title: EHUVEC_deltaVP30WT_48h_1_41

source_name: HUVEC cells, deltaVP30-WT-inoculated, 48h, bioreplicate 1


title: TB-only_Diagnosis_Indonesia_UNPAD16


title: EHUVEC_deltaVP30WT_8h_3_33

source_name: HUVEC cells, deltaVP30-WT-inoculated, 8h, bioreplicate 3


title: EHUVEC_deltaVP30WT_0h_1_26

source_name: HUVEC cells, deltaVP30-WT-inoculated, 0h, bioreplicate 1


title: Tumor-Cocultured CLTX-T-CAR Neutrophils 2

city: West Lafayette

first: Xiaoping

last: Bao

state: Indiana

address: 480 Stadium Mall Drive

department: Chemical Engineering

institute: Purdue University

data_processing:
Casava1.8 software used for basecalling
RNA-seq reads were aligned to the hg19 genome assembly using HISAT2
the RefSeq transcript levels (RPKMs) were quantified using the python script rpkmforgenes.py
Genome_build: hg19
Supplementary_files_format_and_content: tab-delimited text files include RPKM values for each sample

source_name: CLTX-T-CAR hPSC-derived Neutrophils after coculture with tumor

growth_protocol: Day 21 neutrophils were generated from hPSCs by treatment of CHIR99021 (day 0-1), VEGF (day 2-6), SB431542 (day 4-6), SCF (day 6-9), FLT3L (day 6-9), GM-CSF (day 9-12), G-CSF and AM580 (day 15-21).

extract_protocol:
RNA of hPSC-derived CD16+ neutrophils was harvested using Trizol reagent. Clontech SMARTer Stranded Total RNA-Seq Kit was used for the construction of sequencing libraries
RNA libraries were prepared for sequencing using Clontech SMARTer Stranded Total RNA-Seq Kit

treatment_protocol: With or without tumor cell co-culture

value: Hematopoietic

tag: hpsc

value: day 21 from H9 hPSC

value: co-culture with glioblastoma cells


title: Tumor-Cocultured CLTX-T-CAR Neutrophils 1


title: Tumor-Cocultured H9 Neutrophils 2

source_name: hPSC-derived Neutrophils after coculture with tumor


title: TB-DM_Diagnosis_South Africa_SUN155

source_name: Whole Blood


title: EHUVEC_deltaVP30deltamucin_72h_4_24

source_name: HUVEC cells, deltaVP30-delta-mucin-inoculated, 72h, bioreplicate 4


title: H9 antiPSMA-Nanobody CAR Neutrophils

value: without tumor co-culture


title: EHUVEC_deltaVP30deltamucin_72h_2_22

source_name: HUVEC cells, deltaVP30-delta-mucin-inoculated, 72h, bioreplicate 2


title: EHUVEC_deltaVP30WT_24h_2_37

source_name: HUVEC cells, deltaVP30-WT-inoculated, 24h, bioreplicate 2


title: EHUVEC_deltaVP30deltamucin_72h_3_23

source_name: HUVEC cells, deltaVP30-delta-mucin-inoculated, 72h, bioreplicate 3


title: Tumor-Cocultured H9 Neutrophils 1


title: EHUVEC_deltaVP30WT_0h_4_29

source_name: HUVEC cells, deltaVP30-WT-inoculated, 0h, bioreplicate 4


title: EHUVEC_deltaVP30deltamucin_72h_1_21

source_name: HUVEC cells, deltaVP30-delta-mucin-inoculated, 72h, bioreplicate 1


title: EHUVEC_deltaVP30deltamucin_48h_5_20

source_name: HUVEC cells, deltaVP30-delta-mucin-inoculated, 48h, bioreplicate 5


title: H9 J591-CAR Neutrophils 2

source_name: J591-CAR hPSC-derived Neutrophils


title: H9 J591-CAR Neutrophils 1


title: EHUVEC_deltaVP30deltamucin_48h_3_18

source_name: HUVEC cells, deltaVP30-delta-mucin-inoculated, 48h, bioreplicate 3


title: H9 IL13-CAR Neutrophils 2

source_name: IL13-CAR hPSC-derived Neutrophils


title: H9 IL13-CAR Neutrophils 1


title: EHUVEC_deltaVP30deltamucin_48h_4_19

source_name: HUVEC cells, deltaVP30-delta-mucin-inoculated, 48h, bioreplicate 4


title: H9 CLTX-NK-CAR Neutrophils

source_name: CLTX-NK-CAR hPSC-derived Neutrophils


title: H9 Neutrophils 2

source_name: hPSC-derived Neutrophils


title: H9 Neutrophils 1


title: EHUVEC_deltaVP30deltamucin_48h_2_17

source_name: HUVEC cells, deltaVP30-delta-mucin-inoculated, 48h, bioreplicate 2


title: sc Monolayers Pooled Conditions [GEX]

city: Durham

first: Jarrett

last: Bliton

address: 6 Davis Drive

institute: Altis Biosystems

description:
monolayer_filtered_feature_bc_matrix.h5
hash_1+3_processed_adata_cc-regressed_k10.h5ad

data_processing:
[Read Alignment]  single-cell fastq files were aligned to reference transcriptome GRCh38 with the 10X Cell Ranger pipeline (V4.0.0),
[Single cell Downstream Processing]  downstream analysis was performed with scanpy (v1.7.2).
Annotations for cell cycle phase were added following previously published methods in Moor et al 2018. Seurat v2 was used to identify 2585 highly variable genes
[Single cell Quality Filtering parameters]  , In vivo scRNASeq, In vitro scRNASeq
Minimum genes, >500, >1000
Percent mitochondrial reads, <75%, <50%
Minimum counts, >3,000, >5,000
Maximum counts, <50,000, <80,000
[Single cell Dimensional reduction, batch correction,  Leiden clustering, and UMAP visualization ]  Following filtering, read counts were log-transformed and normalized to the median read depth of the dataset. For both single cell sequencing experiments no batch correction was performed as each dataset was analyzed separately. Variability due to read count, percentage of mitochondrial reads and cell cycle phase were regressed out by simple linear regression. Highly variably genes were identified using Seurat v2. 2,585 and 4,186 highly variable genes were used for principal component analysis for the in vivo and in vitro scRNAseq datasets, respectively. Counts for each gene were scaled to have a mean of zero and unit variance. A kNN-graph was contruscted with 10 neighbors was used to calculate leiden clusters for both in vivo and in vitro scRNAseq datasets (In vivo clustering parameters: Leiden resolution = 0.5, num_neighbors = 10, num_pcs = 40; in vitro clustering parameters: Leiden resolution = 0.1, num_neighbors = 10, num_pcs = 15) and EPCAM-negative cells were removed from the in vivo dataset. PAGA was used to initialize UMAP embeddings of leiden clusters.
Regional hashtag deconvolution followed published methods for both scRNAseq datasets. Briefly, raw hashtag read counts were normalized using centered log ratio transformation followed by k-medoid clustering (k=6 medoids)19. Hashtag noise distributions were determined by removing the cluster with highest expression of a specific hashtag, then a negative binomial distribution was fit to the data of the remaining cells. Cells were considered positive for a hashtag if counts for the specific hashtag were above the distribution’s 99th percentile (p<0.01) threshold. Cells positive for multiple hashtags were excluded as likely doublets. Cells called for colon and duodenum were removed from the dataset. For in vitro data, only hashtags 1 and 3 were kept for analysis.  Hashtag 1 and hashtag 3 come from the same dataset and need to be deconvoluted from the same set of 108 fastqs.
[Bulk RNA ]  Integrated fluidic circuits (IFCs) for samples with RIN above 8 were prepared for gene expression and genotyping analysis by using the Advanta™ RNA-Seq NGS Library Prep Kit for the Fluidigm Juno™ and sequenced with the Fluidigm Biomark™ HD system. Gene level expression was obtained through pseudo alignment of reads to human genome GRCh38 using Kallisto (Bray et al., 2016). Expression values for plotting were obtained by TMM normalization across all samples using EdgeR package (Robinson et al., 2010). Sequencing data from each time point were combined and then were normalized to the dataset median and log-transformed. Principal component analysis was done with scikitlearn (v0.24.0).
Genome_build: GRCh38
Supplementary_files_format_and_content: h5 - raw feature counts for each dataset
Supplementary_files_format_and_content: h5ad - final processed and integrated dataset for figure generation and analysis

source_name: Human Jejunum

extract_protocol:
[in vivo scRNAseq]  Whole human intestines were transported to UNC Chapel Hill in ice-cold University of Wisconsin Solution, with tissue dissection beginning within eight hours of cross-clamping. First, fat and connective tissue were trimmed from the donated organs and intestines were subdivided into six regions following measurement. For the small intestine, the proximal 20 cm was deemed Duodenum. Jejunum and ileum were determined through an even split of the remaining small intestine. Two 3x3 cm2 resections were isolated from the center of jejunum and ileum for dissociation.   Resections were incubated in 10 mM NAC at room temperature for 30 min to remove mucus, then tissue was moved to ice-cold Isolation Buffer which consisted of 5.6 mM Na2HPO4, 8.0 mM KH2PO4, 96.2 mM NaCl, 1.6 mM KCl, 43.4 mM Sucrose, 54.9 mM d-sorbitol, and 100 uM Y27632, then washed several times by gently inverting the tubes. Tissues were then incubated in Isolation Buffer with 2 mM EDTA and 0.5 mM DTT, then shaken vigorously to remove crypts. Shakes were repeated several times, checking for crypts and/or villi each time. High-yield small intestinal shakes were pooled to approximate 1:1 villus to crypt tissue by cell mass. Crypts and villi were dissociated to single cells using 4 mg/ml Protease VIII in DPBS + Y27632 on ice for ~45min with trituration via a P1000 micropipette every 10 min. Dissociation was checked on a light microscope then clumps were removed using filtration.   Single cells were washed with DPBS + Y27632 then resuspended in Advanced DMEM/F12 + 1% Bovine Serum Albumin + Y-27632. AnnexinV-APC (1:100) was added for live/dead staining and one TotalSeq Anti-Human Hashtag Antibody (Biolegend Cat Nos. 394631, 394633, 394635, 394637, 394639, & 394641) per condition to allow for tracking all six conditions with a single library preparation. Cells were washed with Advanced DMEM/F12 + 1% BSA +Y27632 then resuspended in the same solution for sorting on a Sony Cell Sorter SH800Z. Cells were gated using forward and backward scatter and AnnexinV to enrich for live single epithelial cells. 25k cells were collected from each separate region, then all regions were combined before sequencing. Library prep was performed with the Chromium Next GEM Single Cell 3’ GEM, Library & Gel Bead Kit v3.1. Sequencing was performed on an Illumina NextSeq 500.
[in vitro scRNAseq]  Media was removed from ISCs expanded on collagen and from AEs on 12-well transwell inserts and washed with 1x DPBS. 1.5mls of 3mM EDTA in PBS was applied to ISCs expanded on collagen and on apical and basal reservoirs of 12-well transwell cultures until most cells were lifted off the collagen or  Transwell surface as determined by visual inspection every 2 minutes under an inverted microscope. EDTA was aspirated using a P1000 micropipette and redistributed over the Transwell surfaced to facilitate detachment of cells from the Transwell at 2-minute intervals until the majority of cells had detached. After the cells detached, 1.5mls of DPBS was applied to each well and rinsed by aspirating and re-applying the EDTA/DPBS. Cells were then aspirated and pelleted by centrifugation at 500g for 5 minutes. The supernatant was removed and replaced with 1ml of 4mg/mL cold protease in DPBS. Cells in cold protease were incubated on ice and pipetted every 2 minutes before visual examination of dissociation under an inverted light microscope. This was repeated until all cells were singlets. Following single cell dissociation, the cold protease was quenched with Advanced DMEM/F12 + 1% FBS. Cells were pelleted as described above and resuspended in Advanced DMEM/F12 +1% FBS.
[scRNAseq]  Single cells were washed with DPBS + Y27632 then resuspended in Advanced DMEM/F12 + 1% Bovine Serum Albumin + Y-27632. AnnexinV-APC (1:100) was added for live/dead staining and one TotalSeq Anti-Human Hashtag Antibody (Biolegend Cat Nos. 394631, 394633, 394635, 394637, 394639, & 394641) per condition to allow for tracking all six conditions with a single library preparation. Cells were washed with Advanced DMEM/F12 + 1% BSA +Y27632 then resuspended in the same solution for sorting on a Sony Cell Sorter SH800Z. Cells were gated using forward and backward scatter and AnnexinV to enrich for live single epithelial cells. 25k cells were collected from each separate region, then all regions were combined before sequencing. Library prep was performed with the Chromium Next GEM Single Cell 3’ GEM, Library & Gel Bead Kit v3.1. Sequencing was performed on an Illumina NextSeq 500.
[bulkRNAseq]  To investigate the dynamic changes in gene expression as ISCs differentiate into AEs in vitro, RNA-seq was performed on human intestinal epithelial monolayers immediately before seeding onto Transwells (D0) and at Days 2, 5, 7, 10, and 11  (D2, D5, D7, D10, D11) of differentiation on Transwells.  N=3 samples were collected from each time point and RNA was extracted using RNAqueous-Micro Total RNA Isolation Kit (AM1931; ThermoFisher) according to manufacturer’s protocols and stored at −80°C. RNA quality was assessed prior to library preparation by using the  Agilent 2100 Bioanalyzer to determine the RNA integrity number (RIN) (citation for RIN).
scRNAseq & BulkRNAseq

value: Intestine

tag: intestinal region

value: Jejunum

tag: transwell

value: mixed sample (EM_Transwell, DM_Transwell)

tag: culture conditions

value: mixed sample (ISCs in EM+Y on Expansion scaffolds; ISCs in EM+Y, transwells until confluent, then DM for 5 days)


title: EHUVEC_deltaVP30deltamucin_24h_3_13

source_name: HUVEC cells, deltaVP30-delta-mucin-inoculated, 24h, bioreplicate 3


title: EHUVEC_deltaVP30deltamucin_24h_5_15

source_name: HUVEC cells, deltaVP30-delta-mucin-inoculated, 24h, bioreplicate 5


title: EHUVEC_deltaVP30deltamucin_24h_4_14

source_name: HUVEC cells, deltaVP30-delta-mucin-inoculated, 24h, bioreplicate 4


title: EHUVEC_deltaVP30deltamucin_8h_5_10

source_name: HUVEC cells, deltaVP30-delta-mucin-inoculated, 8h, bioreplicate 5


title: EHUVEC_deltaVP30deltamucin_24h_1_11

source_name: HUVEC cells, deltaVP30-delta-mucin-inoculated, 24h, bioreplicate 1


title: IMR32_untreated_2_2

city: Gent

first: Laurentijn

last: Tilleman

address: Ottergemsesteenweg 460

country: Belgium

institute: Ghent University

data_processing:
Library strategy: TrAEL-seq
Sequencing data was processed with a custom script (location available from the manuscript) to transfer the first 8bp of a each sequence to the readID of the FastQ file (UMI, unique molecular identifier). In addition, up to and including 3 T bases were removed from the 5'-end following the UMI transfer. The resulting FastQ files were trimmed with Trim Galore (v0.6.5) to remove read-through contamination with the Illumina standard adapter as well as poor base call qualities (cutoff: Phred 20). Trimmed reads were then aligned to the relevant genomes using Bowtie2 (v2.4.1) in --local mode. Following the alignments, reads were de-duplicated taking UMIs as well as mapping position into account, using UmiBam (v0.2.0; https://github.com/FelixKrueger/Umi-Grinder).
Samples were imported into Seqmonk v1.47, truncated to a single base and quantified %reverse reads in 1kb windows
Genome_build: GRCh38
TrAEL report: The file is a .txt file containing TrAEL-seq read polarity values for IMR32 cells either untreated, or treated for 24h with 689.5 nM 3AP or DMSO alone. Reads were then summed in 200 kb running windows spaced every 10 kb, and read polarity was quantified by (Reverse-Forward)/(Reverse+Forward) and subtracted by 50 ; data is an average of two biological replicates.

source_name: IMR32 (Neuroblastoma cell line)

growth_protocol: The IMR32 cells were cultured in a T-25 flask in RPMI 1640 media, containing 10% FBS, 100 IU/mL Penicillin/Streptomycin, 2mM L-Glutamine. IMR32 cells were allowed to adhere for 48h, after which the medium was replaced by fresh medium and the either the 0.1% DMSO control or 3AP treatment (689.5 nM) was added. After 24h of treatment, the supernatant of each flask was collected and added to the trypsinised cells. After centrifugation  (5 minutes at 1200 rpm), the pellet was washed with ice-cold PBS. 1 x 106 cells per condition were washed with L Buffer (100 mM EDTA pH8, 10 mM Tris pH7.5, 20 mM NaCl) and finally re-suspended in 60 µl of the buffer. The cell suspension was warmed for at least 2 minutes at 50°C before adding to 40µl melted and warm CleanCut agarose. The mixture was vortexed for 5s and directly pipetted into a plug mould and left to solidify for 30 min at 4°C. Each plug was transferred to a 2-ml tube containing 500 μl digestion buffer (10 mM Tris HCl (pH 7.5), 100 mM EDTA, 20 mM NaCl, 1% sodium N-lauroyl sarcosine, 0.1 mg/ml Proteinase K) and incubated overnight at 50°C. Plugs were then washed and RNase T1 treated.

extract_protocol:
½ an agarose plug of cells was equilibrated once in 100 µl 1x TdT buffer (NEB) for 30 min at room temperature, then incubated for 2 h at 37°C in 100 µl 1x TdT buffer containing 4 µl 10 mM ATP and 2 µl Terminal Transferase (NEB M0315L). Plugs were rinsed with 1 ml tris buffer (10 mM Tris HCl pH 8.0), equilibrated in 100 µl 1x T4 RNA ligase buffer (NEB) containing 40 µl 50% PEG 8000 for 1 hour at room temperature then incubated overnight at 25°C in 100 µl 1x T4 RNA ligase buffer (NEB) containing 40 µl 50% PEG 8000, 1 µl 10 pM/µl TrAEL-seq adaptor 1 ([App]NNNNNNNNAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTUGCGCAGGCCATTGGCC[BtndT]GCGCUACACTCTTTCCCTACACGACGCT) and 1 µl T4 RNA ligase 2 truncated KQ (NEB M0373L). Plugs were then rinsed with 1 ml tris buffer, transferred to 15 ml tubes and washed three times in 10 ml tris buffer with rocking at room temperature for 1-2 hours each, then washed again overnight under the same conditions. Plugs were equilibrated for 15 min with 1 ml agarase buffer (10 mM Bis-Tris-HCl, 1 mM EDTA pH 6.5), then the supernatant removed and 50 µl agarase buffer added. Plugs were melted for 20 min at 65°C, transferred for 5 min to a heating block pre-heated to 42°C, 1 µl β-agarase (NEB M0392S) was added and mixed by flicking without allowing sample to cool, and incubation continued at 42°C for 1 h. DNA was ethanol precipitated with 25 µl 10 M NH4OAc, 1 µl GlycoBlue, 330 µl of ethanol and resuspended in 10 µl 0.1xTE. 40 µl reaction mix containing 5µl Isothermal amplification buffer (NEB), 3 µl 100 mM MgSO4, 2µl 10 mM dNTPs and 1µl Bst 2 WarmStart DNA polymerase (NEB M0538S) was added and sample incubated 30 min at 65°C before precipitation with 12.5 µl 10 M NH4OAc, 1 µl GlycoBlue, 160 µl ethanol and re-dissolving pellet in 130 µl 1xTE.
The DNA was transferred to an AFA microTUBE (Covaris 520045) and fragmented in a Covaris E220 using duty factor 10, PIP 175, Cycles 200, Temp 11, then transferred to a 1.5 ml tube containing 8 µl pre-washed Dynabeads MyOne streptavidin C1 beads (Thermo, 65001) re-suspended in 300 µl 2xTN (10 mM Tris pH 8, 2 M NaCl) along with 170 µl water (total volume 600 µl) and incubated 30 min at room temperature on a rotating wheel. Beads were washed once with 500 µl 5 mM Tris pH 8, 0.5 mM EDTA, 1 M NaCl, 5 min on wheel and once with 500 µl 0.1x TE, 5 min on wheel before re-suspension in 25 µl 0.1x TE. 3.5 μl NEBNext Ultra II End Prep buffer, 1 µl 1 ng/µl sonicated salmon sperm DNA (this is added as a carrier) and 1.5 μl NEBNext Ultra II End Prep enzyme were added and reaction incubated 30 min at room temperature and 30 min at 65°C. After cooling, 1.25 µl 10 pM/µl TrAEL-seq adaptor 2 ([Phos]GATCGGAAGAGCACACGTCTGAACTCCAGTCUUUUGACTGGAGTTCAGACGTGTGCTCTTCCGATC*T ), 0.5 µl NEBNext ligation enhancer and 15 µl NEBNext Ultra II ligation mix were added and incubated 30 min at room temperature. The reaction mix was removed and discarded and beads were rinsed with 500 µl wash buffer (5 mM Tris pH 8, 0.5 mM EDTA, 1 M NaCl) then washed twice with 1 ml wash buffer for 10 min on wheel at room temperature and once for 10 min with 1 ml 0.1x TE. Libraries were eluted from beads with 11 µl 1x TE and 1.5 µl USER enzyme (NEB) for 15 min at 37°C, then again with 10.5 µl 1x TE and 1.5 µl USER enzyme (NEB) for 15 min at 37°, and the two eluates combined. An initial test amplification was used to determine the optimal cycle number for each library. For this, 1.25 μl library was amplified in 10 µl total volume with 0.4 µl each of the NEBNext Universal and any NEBNext Index primers with 5 µl NEBNext Ultra II Q5 PCR master mix. Cycling program: 98°C 30s then 18 cycles of [98°C 10 s, 65°C 75 s], 65°C 5 min. Test PCR was cleaned with 8 µl AMPure XP beads (Beckman A63881) and eluted with 2.5 µl 0.1x TE, of which 1 µl was examines on a Bioanalyser high sensitivity DNA chip (Agilent 5067-4626). Ideal cycle number should bring final library to final concentration of 1-3 nM, noting that the final library will be 3 cycles more concentrated than the test anyway. 21 µl of library was then amplified with 2 µl each of NEBNext Universal and chosen Index primer and 25 µl NEBNext Ultra II Q5 PCR master mix using same conditions as above for calculated cycle number. Amplified library was cleaned with 40 µl AMPure XP beads (Beckman A63881) and eluted with 26 µl 0.1x TE, then 25 µl of this was again purified with 20µl AMPure XP beads and eluted with 11µl 0.1x TE.

tag: cell type

value: IMR32 (Neuroblastoma cell line)

value: Untreated


title: EHUVEC_deltaVP30deltamucin_8h_4_9

source_name: HUVEC cells, deltaVP30-delta-mucin-inoculated, 8h, bioreplicate 4


title: H9 CLTX-T-CAR Neutrophils

source_name: CLTX-T-CAR hPSC-derived Neutrophils

tag: treatment


title: IMR32_3AP_IC50_24hr_1_5


title: EHUVEC_deltaVP30deltamucin_8h_3_8

source_name: HUVEC cells, deltaVP30-delta-mucin-inoculated, 8h, bioreplicate 3


title: IMR32_untreated_1_1


title: EHUVEC_deltaVP30deltamucin_24h_2_12

source_name: HUVEC cells, deltaVP30-delta-mucin-inoculated, 24h, bioreplicate 2


title: EHUVEC_deltaVP30deltamucin_8h_2_7

source_name: HUVEC cells, deltaVP30-delta-mucin-inoculated, 8h, bioreplicate 2


title: sc Monolayers Pooled Conditions [CSP]

molecule: protein

tag: hashtag

value: mixed sample (hashtag_1, hashtag_3)


title: EHUVEC_deltaVP30deltamucin_48h_1_16

source_name: HUVEC cells, deltaVP30-delta-mucin-inoculated, 48h, bioreplicate 1


title: IMR32_0_1_DMSO_24hr_1_3

value: DMSO


title: EHUVEC_deltaVP30deltamucin_0h_4_4

source_name: HUVEC cells, deltaVP30-delta-mucin-inoculated, 0h, bioreplicate 4


title: IMR32_3AP_IC50_24hr_2_6