title: normoxia_1hr_U2OS_biorep4_Input

first: Angela

last: Simeone

address: 200 Hathersage Rd

institute: Genomic Services QIAGEN

data_processing:
G4-ChIP-seq Fastq files were trimmed from adapters using cutadapt (-m 10 -q 20 -O 3 -a CTGTCTCTTATACACATCT) and aligned to a the human genome hg38 with bwa-mem. Bam files were generated from the alignment with samtools view. Duplicated reads were marked and removed using picard MarkDuplicates (ver: 2.20.3).Peak calling was performed with macs2 over input (ver. 2.1.2) with fdr 0.05 (default). For G4-ChIP-seq, regions confirmed across 2 out of 3 technical replicates have been assigned to each biological replicate. Regions shared by 2 out of 3 biological replicates have been elected as consensus of the cell type and multiIntersectBed was used for this.
Genome_build: hg38
Supplementary_files_format_and_content: narrowPeak: narrowPeak regions obtained with macs2 default options
Supplementary_files_format_and_content: bed: consensus representing genomic regions observed in 2/3 of the experiments available (if 3 replicates: 2 out of 3; if 12 replicates : 8 out of 12; f 6 replicates: 4 out of 6)

source_name: cultured U2OS cells

growth_protocol:
K562 cells were cultured in RPMI 1640 supplemented with 10% of fetal bovine serum (FBS), and were incubated at 37°C in 5% CO2 unless stated otherwise.
K562 cells profiling by G4-ChIP-seq upon DRB, TPL; K562 cells profiling by G4-ChIP-seq, Pol2-ChIP-seq and Atac-seq upon pyPDS, Hypoxia, Hypoxia plus pyPDS or DMSO, Normoxia plus pyPDS or DMSO.U2OS cells (ATCC, HTB-96) were cultured in DMEM (Thermo Fisher, 41966-029) supplemented with 10% of FBS

extract_protocol:
Cell nuclei were extracted and sonicated. G4 containing DNA were respectively isolated with BG4 antibody.
For G4 ChIP libraries, DNA was tagmented and the library was prepared using Nextera library preparation kit (20034197) by following the manufacturer instruction. The library was amplified using Nextera index primers (Illumina, FC-121-1030). For RNA Pol II ChIP-seq, library was prepared by using NEBNext® Ultra™ II DNA Library Prep Kit for Illumina (E7645).
G4 ChIP-seq

treatment_protocol: For hypoxia treatment, cells were incubated at 1% O2, 5% CO2 and 37°C. DRB, triptolide and pyPDS were dissolved in DMSO at a final concentration of 100 µM, 10 µM and 1 µM respectively. For pyPDS stabilisation in hypoxia, cells were exposed to 1% O2 following 1-hour pyPDS treatment.

value: U2OS cells

tag: chip antibody

value: normoxia 21% O2 for 1 hr following 1 hr DMSO (0.1% v/v) treatment


title: normoxia_1hr_U2OS_biorep4_ChIP3

status: Public on Apr 02 2021

type: SRA

city: Manchester

country: United Kingdom

library_source: genomic

molecule: genomic DNA

organism: Homo sapiens

tag: cell line

tag: treatment


title: RNA_ID2_1

city: Chicago

first: Weiguo

last: Cui

state: IL

address: 303 E Chicago Ave

department: Pathology

country: USA

institute: Northwestern University

library_source: transcriptomic

data_processing:
Reads were aligned to the mouse reference genome (mm9) using Tophat (version 2.1.1)
Expression values for each gene were quantified and differential gene expression analysis was carried out using Cuffdiff (Cufflinks version 2.2.2)
Genome_build: mm10
Supplementary_files_format_and_content: genes.read_group_tracking_MIG_E47_ID2 contains the FPKM values for each gene in each replicate

molecule: polyA RNA

organism: Mus musculus

source_name: P14 CD8 T cells

growth_protocol: P14 cells were activated for one day with 100 ng/ml gp33 and 1 ng/ml IL-2. Cells were then spin transduced with 1 ml of either MSCV-IRES-GFP (MIG), MIG-E47, or MIG-ID2 retrovirus, 8 ug/ml polybrene, 10 ng/ml IL-2, and 10 mM HEPES Buffer (Corning) for 90 minutes at 32 ºC at 2,500 rpm. Retrovirus-containing media was then removed and replaced with the original media, followed by an additional one day of activation. P14 cells were then washed with PBS and cultured in 10 ng/ml IL-2 for two days for expansion. CD8+ GFP+ cells were then sorted and prepared for RNA-seq.

extract_protocol:
Total RNA from P14 CD8 T cells transduced with MIG, MIG-E47, or MIG-ID2 was purified from sorted cells using RNeasy Plus Mini Kit (QIAGEN).
mRNA isolation and library preparation were completed using NEBNext Poly(A) mRNA Magnetic Isolation Module and NEBNext Ultra RNA Library Prep Kit for Illumina (NEB).

tag: strain background

tag: cell type

value: P14 CD8 T cells

tag: cell subset

value: CD8+ GFP +

tag: transduction


title: normoxia_1hr_U2OS_biorep4_ChIP2


title: RNA_MIG_1

value: MIG


title: normoxia_1hr_U2OS_biorep4_ChIP1


title: normoxia_1hr_U2OS_biorep3_ChIP3


title: normoxia_1hr_U2OS_biorep3_Input


title: normoxia_1hr_U2OS_biorep3_ChIP2


title: normoxia_1hr_U2OS_biorep3_ChIP1


title: normoxia_1hr_U2OS_biorep1_Input


title: normoxia_1hr_U2OS_biorep1_ChIP3


title: hypoxia_1hr_U2OS_biorep4_Input

value: treated in 1% O2 for 1 hr following 1 hr DMSO (0.1% v/v) treatment


title: normoxia_1hr_U2OS_biorep1_ChIP2


title: normoxia_1hr_U2OS_biorep1_ChIP1


title: hypoxia_1hr_U2OS_biorep1_ChIP2


title: hypoxia_1hr_U2OS_biorep3_ChIP2


title: mimic,rep3

city: Orléans

first: Chantal

last: Pichon

address: Rue Charles Sadron

country: France

institute: Centre de Biophysique Moléculaire, CNRS-UPR4301

data_processing:
Image analysis, base calling and demultiplexing were performed using RTA 2.4.6 and bcl2fastq 2.18.12
Adapter sequences were trimmed with Cutadapt-1.15, only reads longer than 10 bp were kept for analysis
Sequences were aligned to the Human genome  with TopHat2 2.1.1 using default parameters
Uniquely mapped reads were assigned to genes with FeatureCounts- subread v.1.5.2
Differential expression analysis was performed with DESeq2,
Genome_build: Hg 19 gencode v.17
Supplementary_files_format_and_content: Matrix table with normalized abundance measurements for every gene and every sample (output from DESeq2)

source_name: Spontaneously immortalized human keratinocyte cell line

extract_protocol:
Total RNA was isolated from HaCaT cells using mirVanaTM miRNA isolation kit (Ambion, Life Technologies, Cat #AM1560)
Libraries were constructed using the Illumina mRNA Stranded Kit, according to the manufacturer recommandations

value: HaCaT cells

tag: transfection


title: hypoxia_1hr_U2OS_biorep4_ChIP2


title: CITE-seq of leukapheresis-purified, 0.01% digitonin permeabilized, FACS neutrophil-depleted PBMCs, method comparison

city: Seattle

first: Allen Institute

last: For Immunology

state: Washington

address: 615 Westlake Ave N, Seattle, WA

institute: Allen Institute

description:
X066-RP0C1W1_leukopak_perm-cells_cite_200M_rna_counts.h5
X066-RP0C1W1_leukopak_perm-cells_cite_48M_adt_counts.csv.gz
X066-RP0C1W1
The FASTQs are from human subjects and will be provided via dbGAP
OTHER: CITE-seq

data_processing:
Demultiplexing of CITE-seq raw base call files into FASTQ files was performed using 10x Genomics cellranger mkfastq (v.3.0.2).
To perform comparisons between these methodologies, all scRNA-seq and scATAC-seq libraries were downsampled to 200 million FASTQ entries (200M of each sequenced read; ~20,000 reads per expected cell recovered at ~10,000 cell expected recovery), and all ADT libraries were downsampled to 48M reads (Limited by the available read count for 1 experiment; ~4,800 reads per expected cell recovered).
For CITE-seq data, RNA alignment was performed using cellranger (v5.0.0) with the –include-introns parameter against 10x Genomics reference refdata-gex-GRCh38-2020-A. To analyze CITE-seq ADT libraries we used BarCounter to compute the counts of ADT barcodes for each 10x cell barcode
genome_build: Ensembl GRCh38-3.0.0
processed_data_files_format_and_content: Single cell CSVs (Commas separated), and HDF5 file (Hierarchical Data Format).

molecule: total RNA

source_name: peripheral blood mononuclear cells

extract_protocol:
Biological specimens were purchased from BioIVT as cryopreserved PBMCs. All sample collections were conducted by BioIVT under IRB-approved protocols, and all donors sign informed consent forms. PBMCs sourced from BioIVT were isolated using leukapheresis. Following isolation, PBMCs were subjected to RBC lysis, washing, and counting. PBMC aliquots were cryopreserved in Cryostor CS10 (StemCell Technologies, 07930) and stored in vapor phase liquid nitrogen.
CITE-seq library generation was performed using 10x Genomics Single Cell 3’ v3.1 kit, based on the 10x 3’ Gene Expression Library Construction Guide (CG000204 Rev B) and the New York Genome Center Technology Innovation Lab CITE-seq protocol (Version 2019-02-13). Briefly, a Chromium Next GEM Chip G (10x Genomics, 2000177) was placed in a Chromium Next GEM Secondary Holder and 50% Glycerol was dispensed into all unused wells. A barcoding master mix was prepared which consisted of RT Reagent B (10x Genomics, 2000165), Template Switch Oligo (10x Genomics, 3000228), Reducing Agent B (10x Genomics, 2000087), RT Enzyme C (10x Genomics, 2000085). The master mix was added to the sample well, pipette-mixed, and loaded into row 1 of the chip. Chromium Single Cell 3’ v3.1 Gel Beads (10x Genomics, 2000164) were vortexed for 30 seconds and loaded into row 2 of the chip, along with Partitioning Oil (10x Genomics, 2000190) in row 3. A 10x Gasket (10x Genomics, 370017) was placed over the chip and attached to the Secondary Holder. The chip was loaded into a Chromium Single Cell Controller instrument for GEM generation. GEMs were separated into a biphasic mixture through addition of Recovery Agent (10x Genomics, 220016), the aqueous phase was retained and removed of barcoding reagents using Dynabead MyOne SILANE beads. cDNA amplification was performed using the 10x Genomics cDNA Primers (2000089) on a C1000 Touch thermal cycler with 96-Deep Well Reaction Module: 53°C for 45 min, 85°C for 5 min, followed by a hold at 4°C.  To separate ADT and cDNA libraries, we performed a 0.6x bead:sample SPRIselect reagent size-selection. The bead fraction from this separation was retained as the cDNA fraction, and supernatant separation was retained as the ADT-containing fraction. To further process ADTs, we added an additional 1.4x SPRI volumes to the ADT-containing supernatant to a final ratio of 2x SPRI:sample, then retained the bead fraction of this second step. We then repeated a fresh 2x bead:sample cleanup, retaining the bead fraction as the ADT library. ADTs were amplified in a 100 µl reaction consisting of Buffer EB, KAPA HiFi HotStart ReadyMix, SI-PCR-Oligo primer (10 µM Supplementary File 5), and ADT i7 primer (10 µM Supplementary File 5). ADT PCR was performed in a C1000 Touch thermal cycler with 96-Deep Well Reaction Module: 95°C for 3 min, 12 cycles of: 95°C for 20 sec, 60°C for 30 sec, 72°C for 20 sec, followed by a final extension of 72°C for 5 min. SPRIselect reagent cleanups were performed with a 1.6x bead:sample ratio to generate final ADT libraries.cDNA Fragmentation, End-Repair, and A-tailing were performed in a joint reaction containing 25% of cDNA sample as described in the 10x Multiome User Guide. Briefly, cDNA was diluted in Buffer EB (Qiagen, 1014609), and combined with master mix of Fragmentation Buffer (10x Genomics, 2000091) and Fragmentation Enzyme (10x Genomics, 2000090). The reaction was incubated in a C1000 Touch thermal cycler with 96–Deep Well Reaction Module: 4°C start, 32°C for 5 min, 65°C for 30 min, followed by a brief hold at 4°C. Reactions were purified using a dual-sided 0.6x/0.8x bead:sample SPRIselect reagent (Beckman Coulter, B23318) size-selection clean-up. Ligation was performed by mixing each sample with a master mix consisting of Ligation Buffer (10x Genomics, 2000092), DNA Ligase (10x Genomics, 220110), and Adapter Oligos (10x Genomics, 2000094), and incubating in a C1000 Touch thermal cycler with 96–Deep Well Reaction Module for 15 minutes at 20°C, followed by a brief hold at 4°C. Ligation reactions were purified using a 0.8x bead:sample SPRIselect reagent (Beckman Coulter, B23318) clean-up.  Sequencing ready Gene Expression libraries were constructed by amplifying cDNA fragments in a sample indexing PCR consisting of Amp Mix (10x Genomics, 2000047) and Dual Index TT Set A (10x Genomics, 3000431) as described in the 10x Multiome User Guide. Amplification was performed in a C1000 Touch thermal cycler with 96–Deep Well Reaction Module: 98°C for 45 sec, for 14 cycles of: 98°C for 20 sec, 54°C for 30 sec, 72°C for 20 sec, with a final extension of 72°C for 1 min. Final Gene Expression libraries were prepared using a dual-sided 0.6x/0.8x bead:sample SPRIselect reagent (Beckman Coulter, B23318) size-selection clean-up.

value: BioIVT

value: leukapheresis

value: whole cells

value: FACS

value: Dead Cell/Debris/Neutrophil Depletion

value: CITE-seq


title: hypoxia_1hr_U2OS_biorep4_ChIP3


title: scATAC-seq of leukapheresis-purified, 0.01% digitonin permeabilized, FACS neutrophil-depleted PBMCs, method comparison

description:
X066-AP0C1W1_leukopak_perm-cells_atac_200M_atac_filtered_fragments.tsv.gz
X066-AP0C1W1_leukopak_perm-cells_atac_200M_atac_filtered_metadata.csv.gz
X066-AP0C1W1_leukopak_perm-cells_atac_200M_atac_singlecell.csv.gz
X066-AP0C1W1
The FASTQs are from human subjects and will be provided via dbGAP

data_processing:
Binary Base Call (BCL) files were demultiplexed into FASTQ files was performed using 10x cellranger-atac mkfastq (10x Genomics v.1.1.0).
To assess samples at an equal sequencing depth, FASTQ files were downsampled to a uniform total raw read count among compared samples: 2×108 fragments for comparison of nuclei and cells across FACS conditions (Figure 1 and 2); 1.25×108 fragments for optimization experiments due to lower available total read depth after sequencing.
10x cellranger-atac count was used to process sequencing reads by performing adapter trimming and sequence alignment to the GRCh38 (hg38) reference genome (refdata-cellranger-atacGRCh38-1.1.0). The output files fragments.tsv.gz and singlecell.csv were utilized for downstream processing and quality control analysis.
genome_build: Ensembl GRCh38-3.0.0
processed_data_files_format_and_content: Fragment TSVs (Tab separated), Single cell CSVs (Commas separated), and HDF5 file (Hierarchical Data Format).

extract_protocol:
Biological specimens were purchased from BioIVT as cryopreserved PBMCs and Bloodworks NW as freshly drawn whole blood. All sample collections were conducted by BioIVT and Bloodworks NW under IRB-approved protocols, and all donors sign informed consent forms.  PBMCs sourced from BioIVT were isolated using either Ficoll-Paque or leukapheresis. Following isolation, PBMCs were subjected to RBC lysis, washing, and counting. PBMC aliquots were cryopreserved in Cryostor CS10 (StemCell Technologies, 07930) and stored in vapor phase liquid nitrogen.   For fresh blood samples from Bloodworks NW, PBMC processing occurred in-house. Blood tubes were pooled, gently swirled until fully mixed, about 30 times, and diluted with an equivalent volume of room temperature PBS (Thermo Fisher Scientific, 14190235). PBMCs were isolated using one or more Leucosep tubes (Greiner Bio-One, 227290) loaded with 15 mL of Ficoll Premium (GE Healthcare, 17-5442-03) to which a 3 mL cushion of PBS had been slowly added on top of the Leucosep barrier. Diluted whole blood (24-30mL) was slowly added to each tube and spun at 1000×g for 10 minutes at 20°C with no brake (Beckman Coulter Avanti J-15RIVD with JS4.750 swinging bucket, B99516). PBMCs were recovered from the Leucosep tube by quickly pouring all volume above the barrier into a sterile 50 mL conical tube (Corning, 352098). 15 mL cold PBS+0.2% BSA (Sigma, A9576; “PBS+BSA”) was added and the cells were pelleted at 400×g for 5-10 minutes at 4-10°C. The supernatant was quickly decanted, the pellet dispersed by flicking the tube, and the cells washed with 25-50 mL cold PBS+BSA. Cell pellets were combined as needed, the cells were pelleted as before, supernatant quickly decanted, and residual volume was carefully aspirated. PBMCs were resuspended in 1 mL cold PBS+BSA per 15 mL whole blood processed and counted with a ViCell (Beckman Coulter) using VersaLyse reagent (Beckman Coulter, A09777) or with a Cellometer Spectrum Cell Counter (Nexcelom) using ViaStain Acridine Orange/Propidium Iodide solution (Nexcelom, C52-0106-5). PBMCs were cryopreserved in Cryostor10 (StemCell Technologies, 07930) or 90% FBS (Thermo Fisher Scientific, 10438026) / 10% DMSO (Fisher Scientific, D12345) at 5×106 cells/mL by slow freezing in a Coolcell LX (VWR, 75779-720) overnight in a -80°C freezer followed by transfer to liquid nitrogen.
Isolation of nuclei suspensions was performed according to the Demonstrated Protocol: Nuclei Isolation for Single Cell ATAC Sequencing (10x Genomics, CG000169 Rev C). Briefly, 8×105 to 1×106 cells were added to a 1.5 mL low binding tube (Eppendorf, 022431021) and centrifuged (300×g for 5 minutes at 4?) using a swinging bucket rotor (Beckman Coulter Avanti J-15RIVD with JS4.750 swinging bucket, B99516). The supernatant was removed using a vacuum aspirator pipette and the cell pellet was resuspended in 100 µl of chilled 10x Genomics Nuclei Isolation Buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% Tween-20, 0.1 % NP-40 Substitute CAS 9016-45-9 (BioVision 2127-50), 0.01% Digitonin (MP Biomedicals 0215948082), 1% BSA) by pipette-mixing 10 times. Cells were incubated on ice for 3 minutes, followed by dilution with 1 mL of chilled 10x Wash Buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% Tween-20 (BioRad 1610781), 1% BSA) by pipette-mixing 5 times. Nuclei were centrifuged (500×g for 3 minutes at 4?) and the supernatant was slowly removed using a vacuum aspirator pipette. Nuclei were resuspended in chilled 1x Nuclei Buffer (10x Genomics, 2000207) to a target concentration of 3,000 - 6,000 nuclei per µl. Nuclei suspensions were passed through 35 µm Falcon Cell Strainers (Corning, 352235) and counted on a Cellometer Spectrum Cell Counter (Nexcelom) using ViaStain Acridine Orange/Propidium Iodide Staining Solution (Nexcelom, C52-0106-5).  In addition to 10x Nuclei Isolation Buffer (10xNIB), we tested an alternative Nuclei Isolation Buffer (ANIB) as described previously (Mulqueen et al., 2019: 10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% Tween-20, 0.1 % IGEPAL CAS 9002-93-1 (Sigma, I8896), 1x Protease Inhibitor (Roche, 11836170001)). For each buffer, we generated a titration series of detergent concentrations relative to the concentrations described above but did not alter the concentration of other buffer ingredients: 1x, 0.5x, 0.25x, 0.1x for 10xNIB, and 1x and 0.1x for ANIB. The resulting nuclei were imaged using an EVOS M5000 Imaging System (Thermo Fisher Scientific, AMF5000) in transmitted light mode at 40x magnification to visually evaluate nuclear integrity. The 1x 10xNIB, 0.25x 10xNIB, 0.1x 10xNIB, and 1x ANIB were used for 10X scATAC-seq.  For permeabilized cells we prepared a 5% w/v digitonin stock by diluting powdered Digitonin (MP Biomedicals, 0215948082) with 100% DMSO (Fisher Scientific, D12345) and creating 20 µl aliquots which were stored at -20°C. To permeabilize, 1,000,000 cells were added to a 1.5 mL low binding tube (Eppendorf, 022431021) and centrifuged (400×g for 5 minutes at 4°C) using a swinging bucket rotor (Beckman Coulter Avanti J-15RIVD with JS4.750 swinging bucket, B99516). The supernatant was removed using a vacuum aspirator pipette and the cell pellet was resuspended in 100 µl of chilled isotonic Perm Buffer (20 mM Tris-HCl pH 7.4, 150 mM NaCl, 3 mM MgCl2, 0.01% Digitonin) by pipette-mixing ten times. Cells were incubated on ice for 5 minutes, after which they were diluted with 1 mL of isotonic Wash Buffer (20 mM Tris-HCl pH 7.4, 150 mM NaCl, 3 mM MgCl2) by pipette-mixing 5 times. Cells were centrifuged (400×g for 5 minutes at 4°C) using a swinging bucket rotor and the supernatant was slowly removed using a vacuum aspirator pipette. The cell pellet was resuspended in chilled TD1 buffer (Illumina, 15027866) by pipette-mixing to a target concentration of 2,300 - 10,000 cells per µl. Cells were passed through 35 µm Falcon Cell Strainers (Corning, 352235) and counted on a Cellometer Spectrum Cell Counter (Nexcelom) using ViaStain Acridine Orange/Propidium Iodide solution (Nexcelom, C52-0106-5). For optimization, we used varying final digitonin concentrations in the Perm Buffer: 0.01% w/v, 0.05% w/v, 0.1% w/v, and 0.2% w/v. The optimal concentration observed was 0.01% w/v.  scATAC-seq libraries were prepared according to the Chromium Single Cell ATAC v1.1 Reagent Kits User Guide (CG000209 Rev B) with several modifications. 15,000 cells or nuclei were loaded into each tagmentation reaction. Nuclei were brought up to a volume of 5 µl in 1x Nuclei Buffer (10x Genomics, 2000207), mixed with 10 µl of a transposition master mix consisting of ATAC Buffer B (10x Genomics, 2000193) and ATAC Enzyme (Tn5 transposase; 10x Genomics, 2000123). Permeabilized cells were brought up to a volume of 9 µl in TD1 buffer (Illumina, 15027866) and mixed with 6 µl of Illumina TDE1 Tn5 transposase (Illumina, 15027916). Transposition was performed by incubating the prepared reactions on a C1000 Touch thermal cycler with 96–Deep Well Reaction Module (Bio-Rad, 1851197) at 37°C for 60 minutes, followed by a brief hold at 4°C. A Chromium NextGEM Chip H (10x Genomics, 2000180) was placed in a Chromium Next GEM Secondary Holder (10x Genomics, 3000332) and 50% Glycerol (Teknova, G1798) was dispensed into all unused wells. A master mix composed of Barcoding Reagent B (10x Genomics, 2000194), Reducing Agent B (10x Genomics, 2000087), and Barcoding Enzyme (10x Genomics, 2000125) was then added to each sample well, pipette-mixed, and loaded into row 1 of the chip. Chromium Single Cell ATAC Gel Beads v1.1 (10x Genomics, 2000210) were vortexed for 30 seconds and loaded into row 2 of the chip, along with Partitioning Oil (10x Genomics, 2000190) in row 3. A 10x Gasket (10x Genomics, 370017) was placed over the chip and attached to the Secondary Holder. The chip was loaded into a Chromium Single Cell Controller instrument (10x Genomics, 120270) for GEM generation. At the completion of the run,

value: permeabilized cells

value: 0.01% digitonin

value: 10x scATAC-seq


title: hypoxia_1hr_U2OS_biorep1_Input


title: mimic,CTL,rep2

value: negative control (1nM)


title: hypoxia_1hr_U2OS_biorep3_Input

value: U2OS cells


title: hypoxia_1hr_U2OS_biorep3_ChIP1


title: hypoxia_1hr_U2OS_biorep4_ChIP1


title: hypoxia_1hr_U2OS_biorep1_ChIP3


title: hypoxia_1hr_U2OS_biorep1_ChIP1


title: mimic,rep1


title: SAEC_1-2

city: Boston

first: Christopher

middle: J

last: Benway

state: MA

address: 300 Longwood Ave, Enders 510

department: Nephrology

institute: Children's Hospital

data_processing:
NextSeq control software 2.1.0.31 and RTA 2.4.11
The quality of sequencing was evaluated with FastQC and adapter sequences were trimmed with Cutadapt.
Paired-end reads were aligned to human genome build hg19 using Bowtie (v1.0.1) with a maximum insert size of 2000bp.
Reads aligning to the mitochondrial genome; unknown and random chromosomes; and PCR duplicates were removed prior to peak calling using Picard 1.114.
Peaks were called on individual samples and concatenated files combining all technical and biological replicates for each cell type via MACS2 (v2.1.1.20160309) “callpeak” with the following options: ' -g hs -f BAMPE --keep-dup all --call-summits -B'.
Peaks overlapping blacklisted repetitive and known artifact regions as described by ENCODE were removed.
BigWig coverage tracks were generated from MACS2 bedGraphs using bedGraphToBigWig
Genome_build: hg19
Supplementary_files_format_and_content: BED files corresponding to MACS2 narrowPeak format contain peak intervals, scores, significance, summits, and subpeaks.

source_name: small airway epithelial cells

growth_protocol: Normal human bronchial epithelial (NHBE, Lonza) cells were cultured in BEGM Bronchial Epithelial Cell Growth Medium (Lonza). Small airway epithelial cells (SAEC, Lonza) were cultured in SAGM Small Airway Epithelial Cell Growth Medium (Lonza). Lung fibroblasts (LF) and 16HBE14o- cell were cultured in MEM supplemented with 10% FBS. Alveolar type II cells were treated prepared for ATAC-seq immediately after isolation by FACS sorting.

extract_protocol:
Omni-ATAC-seq library preparation was performed as previously described (PMCID: PMC5623106). Airway epithelial cells and cultured lung fibroblasts were grown to ~70% confluency, harvested by trypsinization and washed in cold PBS. Aliquots of 50,000 cells were centrifuged for 5 min at 4°C and 500 × g then resuspended in ATAC-resuspension buffer (RSB) containing NP40, Tween-20, and digitonin for 3 min on ice. ATII cells were aliquoted immediately after FACS sorting and similarly resuspended in RSB. Cells were washed with RSB containing Tween-20 only, centrifuged, and resuspended in transposition mixture containing PBS, Tween-20, digitonin and transposase enzyme (Nextera). Fast-ATAC-seq of 16HBE cells was performed as previously described (PMCID: PMC5042844), wherein the RSB lysis step is omitted and the cells were immediately resuspended in transposase mixture containing only digitonin as detergent. The transposition reaction was incubated at 37°C with shaking at 1000 RPM for 30 min. After transposition, DNA was purified with the MinElute Reaction Clean-up Kit (QIAGEN) and eluted in 20 μL elution buffer. The libraries were pre-amplified as previously described for 5 cycles and an analytical qPCR was performed to determine the additional number of amplification cycles required to ensure sufficient material and limit PCR duplication. The final PCR amplification was performed, and libraries were again purified and eluted in 20 uL H20. Prior to sequencing the libraries were quantified by Agilent D1000 ScreenTape on the Agilent 2200 TapeStation system and SYBR qPCR to assess functional concentration.
Transposase treatment and library construction was performed using the Nextera DNA Library Preparation Kit (Illumina, FC-121-1030) and custom ordered Nextera PCR primers as described (PMCID: PMC3959825).

tag: method

value: Omni-ATAC-seq

tag: sample type

value: cryopreserved cells cultured in small airway growth media


title: LF_1-1

source_name: lung fibroblast cells

value: primary human lung cell FACS sorted from cadaver tissue and cultured in full media


title: ATII-3-1

source_name: alveolar type II pneumocytes

value: primary human lung cell FACS sorted from cadaver tissue


title: SAEC_2-1


title: SAEC_2-2


title: mimic,CTL,rep1


title: 16HBE_F1-2

source_name: 16HBE14o- bronchial epithelial cell line

value: Fast-ATAC-seq

value: cell line cultured in full media


title: 16HBE_F1-1


title: SAEC_1-1


title: hypoxia_1hr_U2OS_biorep3_ChIP3

value: treated in 1% O2 for 1 hr following 1 hr DMSO (0.1% v/v) treatment


title: ATII-1-1


title: U937 cells, Control_1 (ATAC-seq)

city: Stanford

first: Purvesh

last: Khatri

state: California

address: 279 Campus Dr. Beckman Center B235A

department: BMIR

institute: Stanford University

description: Control plasmid transduced U937 cell line 1

data_processing:
All data processing was carried out using the PEPATAC pipeline (http://code.databio.org/PEPATAC/) version 0.8.6
In summary: Adapter trimming was performed by Skewer, followed by QC analysis with FastQC
Reads were aligned to human repeats (https://github.com/databio/ref_decoy) and mitochondrial DNA (http://www.ncbi.nlm.nih.gov/nuccore/251831106) using bowtie2 with the following parameters: -p 30 -k 1 -D 20 -R 3 -N 1 -L 20 -i S,1,0.50
Remaining unaligned reads were aligned to the human genome (GRCh38) using bowtie2 with the following parameters: -p 30 --very-sensitive -X 2000
Duplicates were removed using Picard tools with the following parameters: VALIDATION_STRINGENCY=LENIENT. Peaks were then called using MACS2 callpeak using the following parameters: -f BAMPE -q 0.01 --call-summits --nomodel --keep-dup all. The comparison/control (-c) parameter was supplied with either the control sorted, deduplicated, and aligned BAM files or the ΔNSP sorted, deduplicated, and aligned BAM files depending on the downstream analysis.
Genome_build: GRCh38
Supplementary_files_format_and_content: *_peaks.narrowPeak files are the narrowPeak file outputs from MACS2 (TSVs). Columns are: 'chromosome', 'start', 'end', 'peak name', 'int_neglogqval', '', 'FoldChange'

source_name: Monocytic cell line

growth_protocol: U937 cells were cultured in RPMI1640 (Gibco) supplemented with 10% fetal bovine serum (ATCC), glutamine (Gibco), and penicillin/streptomycin and selected with 500ug/ml geneticin

extract_protocol:
ATAC-seq was performed at Active Motif (Carlsbad, CA)
DNA libraries were prepared for sequencing using standard Illumina protocols

treatment_protocol: U937 cells were treated with 200 units/ml Dnase in HBSS (Invitrogen) and incubated at 37C for 30 mins. Cells were then washed with PBS and cryopreserved in media with 5% DMSO.

value: genetically modified U937

tag: genetic modification

value: Control plasmids

tag: genotype

value: control


title: U937 cells, Control_3 (ATAC-seq)

description: Control plasmid transduced U937 cell line 3


title: NHBE_1-1

source_name: bronchial/tracheal epithelial cells

value: cryopreserved cells cultured in bronchial epithelial growth media


title: mimic,CTL,rep3

value: HaCaT cells


title: mimic,rep2


title: 16HBE_O1-1


title: 16HBE_O1-2


title: 206

city: New York

first: Sander

middle: M

last: Houten

state: NY

address: 1425 Madison Avenue, Box 1498

department: Genetics and Genomic Sciences

institute: Icahn School of Medicine at Mount Sinai

data_processing:
Illumina HiSeq2500 for base caling
Read alignment. STAR (release 2.4.0 g1)
gene-level read quantification via featureCounts
Filtering raw reads: 1 cpm in at least 2 samples
Normalization via calcNormFactors of library edgeR
Genome_build: mouse genome mm10 (GRCm38.75)
Supplementary_files_format_and_content: text file containing filtered, normalized counts per million reads according to normalization factors for Ehhadh WT and Ehhadh KO samples

source_name: liver

extract_protocol:
RNA was isolated using QIAzol lysis reagent followed by purification using the RNeasy kit (Qiagen)
mRNA-focused cDNA libraries were generated using Illumina reagents (polyA capture), and samples were run on an Illumina HiSeq 2500 sequencer to yield a read depth of approximately 56 million 100 nucleotide single-end reads per samples.

tag: strain

tag: tissue

value: liver

value: L-AC


title: 255


title: 153


title: 251

value: wt


title: 197


title: U937 cells, Control_2 (ATAC-seq)

description: Control plasmid transduced U937 cell line 2


title: 135


title: 107


title: 100


title: 66

value: Saline


title: ATII-2-1


title: U937 cells, ΔNSPs_3 (ATAC-seq)

description: Delta-NSPs plasmid transduced U937 cell line 3

value: ΔNSPs

value: depleted of CTSG, ELANE, and PRTN3 simultaneously (deltaNSPs)


title: NHBE_1-2

value: Omni-ATAC-seq


title: U937 cells, ΔNSPs_1 (ATAC-seq)

description: Delta-NSPs plasmid transduced U937 cell line 1


title: 252


title: 99


title: 109


title: 212


title: 43


title: 98


title: U937 cells, ΔNSPs_2 (ATAC-seq)

description: Delta-NSPs plasmid transduced U937 cell line 2


title: 204


title: 196


title: 52


title: 84


title: 244


title: 112


title: OPC_WT4

city: Corbeil-Essonnes

first: Hélène

last: Polvèche

address: 28, Rue Henri Desbruères

institute: I-Stem

description: replicate 4

data_processing:
RNAseq libraries were sequenced on a HiSeq2500, Illumina (paired-end mode)
Mapping & QC-mapped with Tophat2 (2.0.13) & Samtools (0.1.19)
Filtering with PrinSeq (0.20.4)
RNA-seq data were analyzed using FaRLine with the FasterDB database (Benoit-Pilven et al., 2018; Mallinjoud et al., 2014).  The differential gene expression (DEG) between conditions was calculated with DESeq2.
Genome_build: hg19
Supplementary_files_format_and_content: excel files containing splicing events differentially observed (events : acceptor, donor, mutually exclusive, multi exon skipping) and DEGs

source_name: Oligodendrocyte precursor

growth_protocol:
Mouse cortices were mechanical dissociated in the presence of trypsin and DNase. Cortical astrocytes were collected from newborn mice (P1), and cultured in low-glucose DMEM supplemented with 10% FBS and 0.05 mg/ml gentamycin. Cortical neurons were collected at E16.5, and cultured in Neurobasal medium supplemented with B27, antibiotic/antimycotic, 0.5 mM L-glutamine and 5 µM AraC (in the absence of FBS). Primary cells were plated in poly-D-lysine/laminin-coated dishes and cultured for 14 days prior to RNA collection. We followed a primary cell isolation procedure from mouse brain cortices, which allowed us to establish cell cultures of enough purity to study intrinsic omics changes in neurons, astrocytes and oligodendrocytes with minimal cross contamination between cell types (Braz et al., Methods Mol Biol. 2020;2056:217-240. doi: 10.1007/978-1-4939-9784-8_14).
Cortical neurons were collected at E16.5, and cultured in Neurobasal medium supplemented with B27, antibiotic/antimycotic, 0.5 mM L-glutamine and 5 µM AraC (in the absence of FBS). Oligodendrocyte precursor cells (OPCs) were isolated from neonatal pups, using column affinity isolation method with antibodies against a specific OPC marker (PDGFRa) to purify these cells. OPC were directly collected for analysis at this undifferentiated stage, while the rest were differentiated in vitro for 2 weeks to obtain matching populations of differentiated oligodendrocytes (OL). The column flow-through, containing mainly astrocytes, was cultured for 2 weeks in astrocyte medium, to further select for this cell type, eliminate other contaminating cells and allow full differentiation.

extract_protocol:
RNA extraction was performed with the RNeasy Mini kit (QIAGEN; 74104) following the manufacturer’s protocol, including a DNAse digestion step (RNase-Free DNase Set; QIAGEN; 79254) after the first wash with RW1 buffer. RNA concentration was assessed using the NanoDrop (Thermo Scientific) and RNA quality was verified by electrophoresis on an agarose gel.
TruSeq Stranded mRNA (Illumina)

value: Primary oligodendrocyte precursors

value: WT


title: 243


title: 54


title: OPC_WT3

description: replicate 3


title: OPC_WT2

description: replicate 2


title: OPC_SXL4

description: replicate 4

value: DMSXL


title: OPC_WT1

description: replicate 1


title: OPC_SXL2

description: replicate 2


title: OPC_SXL1

description: replicate 1


title: OL_WT4

description: replicate 4

source_name: Oligodendrocyte

value: Primary mature oligodendrocytes


title: OL_WT2

description: replicate 2


title: OPC_SXL3

description: replicate 3


title: OL_WT3

description: replicate 3


title: OL_SXL3

description: replicate 3


title: OL_SXL4

description: replicate 4


title: OL_WT1

description: replicate 1


title: OL_SXL2

description: replicate 2


title: OL_SXL1

description: replicate 1


title: Neurons_WT5

description: replicate 4

source_name: Neuron

value: Primary neurons


title: Neurons_WT4

description: replicate 3


title: Neurons_SXL4

description: replicate 3


title: Neurons_SXL5

description: replicate 4


title: Neurons_SXL3

description: replicate 2


title: Astro_WT4

description: replicate 3

source_name: Astrocyte

value: Primary astrocytes


title: Neurons_WT1

description: replicate 1


title: Neurons_WT3

description: replicate 2


title: Astro_SXL5

description: replicate 4


title: Astro_WT5

description: replicate 4


title: Astro_SXL4

description: replicate 3


title: Astro_SXL2

description: replicate 2


title: Astro_SXL1

description: replicate 1


title: Neurons_SXL1

description: replicate 1


title: Astro_WT2

description: replicate 2


title: Astro_WT1

description: replicate 1


title: Hi-C – ESC rep 3

city: Scarborough

first: Guillaume

last: Filion

state: Ontario

address: 1265 Military Trail

department: Biological Sciences

country: Canada

institute: UTSC

description:
SSEA1+, P-RFP+, X-GFP+, G1
Hi-C_ES_rep_3.hic

data_processing:
All reads from the PaX hybrid cell line were disambiguated and mapped in two steps. First, each read was mapped in the reference genome of 129S1/SvImJ, and independently in the reference genome of CAST/EiJ (both genomes are available from the Wellcome Trust Sanger Institute ftp://ftp-mouse.sanger.ac.uk/REL-1504-Assembly/) using BWA-MEM version 0.7.17 (with options -L 500 for ATAC-Seq and -t4 -P -k17 -U0 -L0,0 -T25 for Hi-C). In each case, the resulting SAM files were processed by samtools version 1.8 with the fixmate option to fill in mate coordinates. Each read was then assigned to a single genome and the coordinates were lifted over to the reference mm10 (Mus musculus genome of strain C57BL/6J) with custom Python scripts available for download at http://github.com/gui11aume/asmap. Briefly, genome assignment was carried out as follows: if the mapping quality of the read was 0 in each genome, the read was considered unmapped and no assignment was performed; otherwise, the read was assigned to the genome with the best alignment score (field AS:i from the output of BWA-MEM). When both alignment scores were equal, the genome of origin was considered ambiguous and no assignment was performed (but the coordinates of the read were still lifted over to mm10). Lift over to mm10 was also performed with custom Python scripts using the positions of the indels of 129S1/SvImJ and CAST/EiJ relative to C57BL/6J. The positions of the indels relative to the reference strain C57BL/6J (mm10) were downloaded from ftp://ftp-mouse.sanger.ac.uk/REL-1505-SNPs_Indels/strain_specific_vcfs.
For RNA-Seq, reads were disambiguated as explained above, and mapped with STAR (standard options) using the Ensembl mouse genome annotation (GRCm38.78). Gene expression was quantified with the STAR option --quantMode GeneCounts. Batch effects were removed using the ComBat function from the sva R package (v.3.22). Sample scaling and statistical analysis were performed with the R package DESeq2, and the log2(vsd) (variance stabilized DESeq2) counts were used for further analysis. Standard TPM (transcripts per million) values were used as an absolute measure of gene expression. From a list of 806 protein-coding genes on the X-chromosome, we masked genes with insufficient sequence polymorphisms, leaving 558 genes. Moreover, we removed genes where expression from the castaneus X chromosome was below the 25th percentile, leaving 335 genes passing quality criteria. The allelic expression ratio was calculated by computing the proportion of reads from the musculus X chromosome for each gene.
For ATAC-Seq, mapping and disambiguation were performed as explained above. Peak calling was performed using Zerone, with option “make atac” at compile time. The ATAC-seq profiles were discretized with option --no-mock. In the process, the four replicates of each time point were merged into a single discretized profile of resolution 300 bp. Only the windows with a confidence score above 99.9% were considered to be ATAC-Seq peaks.
For Hi-C, mapping and disambiguation were performed as explained above, and only the read pairs with at least one end mapped unequivocally were kept for further analyses (i.e., with mapping quality greater than 0 and with a non-ambiguous chromosome of origin). The files were split by genome of origin in order to obtain separate maps for the castaneus and musculus genomes. Duplicate reads were removed using samtools and the contacts were summed by bins of 50 kbp to produce unnormalized Hi-C matrices.
Genome_build: mm10

source_name: embryonic stem cell

growth_protocol: Mouse embryonic stem cells (ESCs) were cultured on 0.2% gelatin-coated dishes in DMEM (Thermo Fisher Scientific, 31966021), supplemented with 10% FBS (ES-qualified, Thermo Fisher Scientific, 16141079) 1,000 U/ml LIF (ORF Genetics, 01-A1140-0100), 1 mM Sodium Pyruvate (Thermo Fisher Scientific, 11360070), 1x MEM Non-Essential Amino Acids Solution (Thermo Fisher Scientific, 11140050), 50U/ml penicillin/streptomycin (Ibian Tech, P06-07100) and 0.1 mM 2-mercaptoethanol (Thermo Fisher Scientific, 31350010). Cells were incubated at 37°C with 5% CO2. The medium was changed every day and cells were passaged using 0.05% Trypsin-EDTA (Thermo Fisher Scientific, 25300054). Cells were monthly tested for mycoplasma contamination using PCR.

extract_protocol:
For RNA-Seq and ATAC-Seq, cells were dissociated using Accutase (Thermo Fisher Scientific, 00-4555-56) (for NPCs), 0.05% Trypsin-EDTA (Thermo Fisher Scientific, 25300054) (for ESCs) or 0.25% Trypsin-EDTA (Thermo Fisher Scientific, 25200056) (for iPSCs) and then stained with SSEA-1 eFluor 660 (Thermo Fisher Scientific, 50-8813-42) for 15 min at 4°C. Cells were washed once with 0.5% BSA in PBS and then FACS sorted using a BD FACSAria II SORP or a BD Influx.  RNA was extracted using the RNeasy Plus Mini Kit (Qiagen, 74136) or RNeasy Micro Kit (Qiagen, 74004) and quantified by Nanodrop.  For ATAC-Seq, 50,000 FACS purified cells were resuspended in 50 μl cold lysis buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.01% Digitonin, 0.1% Tween-20, 0.1% IGEPAL CA-630). After 3 min the lysis was washed out using 1 ml cold lysis buffer containing Tween-20, but no Digitonin or IGEPAL CA-630. Cells were centrifuged for 10 min at 500 rcf and 4°C, supernatant was removed and nuclei were resuspended in 50 μl transposition reaction mix (25 μl Tn5 Transposase buffer (Illumina, 15027866), 2.5 μl Tn5 transposase (Illumina, 15027865), 16.5 µl PBS, 0.01% Digitonin, 0.1% Tween-20, 5 μl nuclease-free water) and incubated at 37°C for 45 min with 1000 RPM mixing. DNA was isolated using the MinElute PCR Purification Kit (Qiagen, 28004).  For Hi-C, cells were dissociated using Accutase (Thermo Fisher Scientific, 00-4555-56) (for NPCs), 0.05% Trypsin-EDTA (Thermo Fisher Scientific, 25300054) (for ESCs) or 0.25% Trypsin-EDTA (Thermo Fisher Scientific, 25200056) (for iPSCs) and then stained with SSEA-1 eFluor 660 (Thermo Fisher Scientific, 50-8813-42). iPSCs were additionally MACS sorted using Anti-SSEA-1 (CD15) MicroBeads (Miltenyi Biotech, 130-094-530). Cells were then fixed for 10 min at room temperature with freshly prepared 1% formaldehyde in PBS (Sigma-Aldrich, F8775-4X25ML) and the reaction then quenched by addition of 0.2M glycine (NZYTech, MB01401). 1x106 cells/ml were permeabilized using 0.1% saponin (Sigma-Aldrich, 47036-50G-F). 10 µg/ml DAPI (Thermo Fisher Scientific, D1306) and 100 µg/ml RNase A (Thermo Fisher Scientific, EN0531) were added and samples incubated for 30 min at room temperature protected from light with slight agitation. After washing once with cold PBS, samples were resuspended in cold 0.5% BSA in PBS at a concentration of 1x107 cells/ml and immediately FACS purified using a BD FACSAria II SORP or a BD Influx. After FACS sorting, dry cell pellets were snap-frozen in dry ice and stored at -80°C.
RNA-Seq libraries were prepared using the TruSeq Stranded Total RNA Library Preparation Kit (Illumina, 20020597) followed by paired-end sequencing (2 x 125 bp) on an Illumina HiSeq 2500.  ATAC-Seq library amplification was performed by two sequential PCR reactions (8 and 4-7 cycles, respectively) using the NEBNext High Fidelity PCR Master Mix (New England Biolabs, M0541S). DNA was then double-size selected using 0.5x and 1.5x Agencourt AMPure XP beads (Beckman, A63880) to isolate fragments between 100 bp and 1 kb. Library quality was assessed on a Bioanalyzer, followed by paired-end sequencing (2 x 125 bp) on an Illumina HiSeq 2500.  For Hi-C, one million cells purified for G0G1 were used as starting materials. Cells were lysed using 250 μl cold lysis buffer (10 mM Tris-HCl pH 8, 10 mM NaCl, 0.2% IGEPAL CA-630) supplemented with 50 µl protease inhibitor cocktail (Sigma-Aldrich, P8340-1ML). Cells were digested with 100 U MboI (New England Biolabs) and incubated for 2 hours at 37°C under rotation, followed by the addition of another 100U for 2 hours and another 100U before overnight incubation. The next day a final 100U were added and incubated for 3 hours. After fill-in with biotin-14-dATP (Thermo Fisher Scientific, 19524016), ligation was performed with 10,000 U T4 DNA Ligase (New England Biolabs, M0202M) overnight at 24°C under rotation. After de-crosslinking, DNA was purified using ethanol precipitation and sonicated to an average size of 300–700 bp with a Bioruptor Pico (Diagenode; seven cycles of 20 s on and 60 s off). Ligation products containing biotin-14-dATP were pulled-down using Dynabeads MyOne Streptavidin T1 beads (Thermo Fisher Scientific, 65601) and end-repaired and A-tailed using the NEBNext End Repair/dA-Tailing Module (New England Biolabs, E6060S and E6053S). Libraries were amplified using the NEBNext High Fidelity PCR Master Mix and NEBNext Multiplex Oligos for Illumina (New England Biolabs, M0541S and E7335S) for 8 cycles and size-selected with 0.9x Agencourt AMPure XP beads. Library quality was assessed on a Bioanalyzer.

treatment_protocol: Neural Precursor Cell Differentiation: ESCs were differentiated to neural precursor cells (NPCs) as described previously (Abranches et al., 2009). ESCs were seeded at a density of 2.75x105 cells/cm2 in N2B27 (50% DMEM/F12 (Thermo Fisher Scientific, 21041025), 50% Neurobasal medium (Thermo Fisher Scientific, 12348017), 1x N2 (Thermo Fisher Scientific, 17502048), 1x B27 (Thermo Fisher Scientific, 12587001)) supplemented with 0.4 µM PD0325901 (Selleck Chemicals, S1036-5mg), 3 μM CHIR99021 (Sigma-Aldrich, SML1046-5MG) and 1,000 U/ml LIF (ORF Genetics, 01-A1140-0100). 24 hours later, cells were dissociated using Accutase (Thermo Fisher Scientific, 00-4555-56) and plated at 2.95x104 cells/cm2 in RHB-A (Takara Bio, Y40001) on 0.2% gelatin-coated T75 flasks, changing media every other day. On days 6 and 8, media was supplemented with 10 ng/ml EGF (R&D Systems, 236-EG-200) and 10 ng/ml bFGF (Thermo Fisher Scientific, 13256029) and additionally with 10µM ROCK inhibitor (Sellekchem, S1049) on day 8. On day 9, cells were dissociated using Accutase (Thermo Fisher Scientific, 00-4555-56) and SSEA1 expressing cells were removed by MACS sorting using Anti-SSEA-1 (CD15) MicroBeads (Miltenyi Biotech, 130-094-530). To completely remove cells that hadn’t undergone XCI, cells were stained with SSEA-1 eFluor 660 (Thermo Fisher Scientific, 50-8813-42) for 15 min at 4°C, washed once with 0.5% BSA in PBS and then SSEA1-/Nanog-RFP-/X-GFP- cells were FACS purified using a BD FACSAria II SORP or a BD Influx (BD Biosciences) at a maximum flow rate of 4,000 ev/s to improve survival. FACS sorted cells were seeded at 3.5x105 cells/cm2 on 0.2% gelatin-coated dishes in RHB-A, supplemented with EGF, FGF, and ROCKi. The medium was changed daily until day 12 when cells reached 100% confluency.  Reprogramming of Neural Precursor Cells: Reprogramming of day 12 neural precursor cells was induced by the addition of 1 mg/ml doxycycline (Tocris, 4090/50) and 25 mg/ml L-ascorbic acid (Sigma-Aldrich, A7506-25G) to the NPC medium (RHB-A supplemented with EGF and FGF). 24 hours later, cells were dissociated using Accutase and seeded on irradiated mouse embryonic fibroblasts (iMEF) in ESC medium containing 15% FBS and supplemented with 1 mg/ml doxycycline and 25 mg/ml L-ascorbic acid. The medium was changed every other day. To isolate iPSC, SSEA1+/Nanog-RFP+/X-GFP+ cells were isolated using FACS at day 10 of reprogramming and re-plated on 0.2% gelatin-coated plates in ESC medium and kept in doxycycline free conditions for 5-6 days.

value: hybrid 129S1/SvImJ, CAST/EiJ

value: embryonic stem cell


title: Hi-C – ESC rep 2

description:
SSEA1+, P-RFP+, X-GFP+, G1
Hi-C_ES_rep_2.hic


title: Hi-C – ESC rep 1

description:
SSEA1+, P-RFP+, X-GFP+, G1
Hi-C_ES_rep_1.hic


title: Sorted CD8 Effector T-cell fraction, Healthy Donor 1

type: RNA

city: Rotterdam

first: Stefan

middle: J.

last: Erkeland

state: Zuid-Holland

address: Dr. Molewaterplein 40

department: Department of Immunology

country: Netherlands

institute: Erasmus University Medical Center

description: Microarray gene expression data from Sorted CD8 Effector T-cell fraction form Healthy Donor 11

hyb_protocol: All cDNA was hybridized to Affymetrix HG-U133 Plus 2.0 GeneChip arrays (containing 54,675 probe sets) according to the Affymetrix GeneChip 3’ IVT Express Kit user manual (Affymetrix)

scan_protocol: GeneChips were scanned using the Affymetrix GeneChip Sanner 3000

data_processing: The data was analyzed by the Affymetrix GeneChip Command Console Scan Control Software. By using Algorithm: Statistical. Quality of the data was checked with the R package “affyQCReport”. Statistically different probesets were calculated with Analysis of Variance (ANOVA) and Significance Analysis of Microarrays (SAM).

label: Biotin

source_name: Healthy Donor, Peripheral Blood Mononuclear Cells

label_protocol: RNA was reverse transcribed into cDNA, purified, fragmented and biotinylated as described previously (Supplementary Methods).

growth_protocol: Peripheral blood mononuclear cells were isolated from healthy donors or patiens by Ficoll-Paque density seperation

extract_protocol: RNA was isolated from mononuclear cells using the Qiagen RNeasy mini kit according to manufacturer's protocols and RNA integrity was checked according to the Agilent RNA 6000 Nano Assay Protocol.

treatment_protocol: CD4+ T cells were labeled with anti-CD3-PerCP-Cy5.5 and anti-CD4-PE-Cy7 and CD8+ T cells were labeled with anti-CD3-PerCP-Cy5.5 and anti-CD8-APC-H7. Naïve T cells (CD27+ and CD45RA+), memory (CD27+ and CD45RO+ and effector (CD27- and CD45RA+) T cells subsets were labeled with, anti-CD45RA-PE, anti-CD27-APC, anti-CD45RO-FITC, anti-CD27-APC and anti-CD45RA-PE antibodies. Cells fractions were sorted on a FACS Aria II cell sorter. When the tumor load of a patient was below 85%, cells were purified using cell sorting  on a FACSAria III (BD Biosciences) based on the CD4 and CD8 expression on T-PLL cells.

value: Peripheral Blood Mononuclear Cells

tag: disease state

value: healthy

tag: cell fraction

value: Sorted CD8 Effector T-cell fraction


title: Sorted CD8 Naive T-cell fraction, Healthy Donor 2

description: Microarray gene expression data from Sorted CD8 Naive T-cell fraction form Healthy Donor 8

value: Sorted CD8 Naive T-cell fraction


title: Sorted CD8 Memory T-cell fraction, Healthy Donor 2

description: Microarray gene expression data from Sorted CD8 Memory T-cell fraction form Healthy Donor 10

value: Sorted CD8 Memory T-cell fraction


title: Sorted CD4 Effector T-cell fraction, Healthy Donor 1

description: Microarray gene expression data from Sorted CD4 Effector T-cell fraction form Healthy Donor 5

value: Sorted CD4 Effector T-cell fraction


title: Sorted CD8 Effector T-cell fraction, Healthy Donor 2

description: Microarray gene expression data from Sorted CD8 Effector T-cell fraction form Healthy Donor 12


title: Sorted CD4 Memory T-cell fraction, Healthy Donor 2

description: Microarray gene expression data from Sorted CD4 Memory T-cell fraction form Healthy Donor 4

value: Sorted CD4 Memory T-cell fraction


title: Hi-C – D5 P-RFP+ rep 3

description:
reprogramming day 5; SSEA1+, P-RFP+, X-GFP-, G1
Hi-C_5NN_rep_3.hic

source_name: neural progenitor cell

value: induced pluripotent stem cell


title: Sorted CD4 Naive T-cell fraction, Healthy Donor 2

description: Microarray gene expression data from Sorted CD4 Naive T-cell fraction form Healthy Donor 2

value: Sorted CD4 Naive T-cell fraction


title: Hi-C – D5 P-RFP+ rep 4

description:
reprogramming day 5; SSEA1+, P-RFP+, X-GFP-, G1
Hi-C_5NN_rep_4.hic


title: Sorted CD4 Memory T-cell fraction, Healthy Donor 1

description: Microarray gene expression data from Sorted CD4 Memory T-cell fraction form Healthy Donor 3


title: T-PLL Sample, Patient 25

description: Microarray gene expression data from T-PLL patient 25

source_name: T-PLL Patient, Peripheral Blood Mononuclear Cells

value: T-PLL


title: Hi-C – ESC rep 4

description:
SSEA1+, P-RFP+, X-GFP+, G1
Hi-C_ES_rep_4.hic


title: Sorted CD8 Naive T-cell fraction, Healthy Donor 1

description: Microarray gene expression data from Sorted CD8 Naive T-cell fraction form Healthy Donor 7


title: T-PLL Sample, Patient 24

description: Microarray gene expression data from T-PLL patient 24


title: Sorted CD8 Memory T-cell fraction, Healthy Donor 1

description: Microarray gene expression data from Sorted CD8 Memory T-cell fraction form Healthy Donor 9


title: Sorted CD4 Effector T-cell fraction, Healthy Donor 2

description: Microarray gene expression data from Sorted CD4 Effector T-cell fraction form Healthy Donor 6


title: Sorted CD4 Naive T-cell fraction, Healthy Donor 1

description: Microarray gene expression data from Sorted CD4 Naive T-cell fraction form Healthy Donor 1


title: T-PLL Sample, Patient 22

description: Microarray gene expression data from T-PLL patient 22


title: T-PLL Sample, Patient 21

description: Microarray gene expression data from T-PLL patient 21


title: Hi-C – D5 P-RFP+ rep 2

description:
reprogramming day 5; SSEA1+, P-RFP+, X-GFP-, G1
Hi-C_5NN_rep_2.hic


title: Hi-C – D5 P-RFP+ rep 1

description:
reprogramming day 5; SSEA1+, P-RFP+, X-GFP-, G1
Hi-C_5NN_rep_1.hic


title: T-PLL Sample, Patient 23

description: Microarray gene expression data from T-PLL patient 23


title: Hi-C – NPC rep 4

description:
SSEA1-, P-RFP-, X-GFP-, G1
Hi-C_NP_rep_4.hic

value: neural progenitor cell


title: T-PLL Sample, Patient 17

description: Microarray gene expression data from T-PLL patient 17


title: Hi-C – NPC rep 2

description:
SSEA1-, P-RFP-, X-GFP-, G1
Hi-C_NP_rep_2.hic


title: T-PLL Sample, Patient 19

description: Microarray gene expression data from T-PLL patient 19


title: RNA-seq – ESC rep 4

description:
SSEA1+ P-RFP+, X-GFP+
RNA-seq_ES.txt


title: Hi-C – NPC rep 1

description:
SSEA1-, P-RFP-, X-GFP-, G1
Hi-C_NP_rep_1.hic


title: T-PLL Sample, Patient 20

description: Microarray gene expression data from T-PLL patient 20


title: RNA-seq – ESC rep 2

description:
SSEA1+ P-RFP+, X-GFP+
RNA-seq_ES.txt


title: RNA-seq – iPSC rep 4

description:
reprogramming day 15/16; SSEA1+, P-RFP+, X-GFP+
RNA-seq_iPS.txt

source_name: induced pluripotent stem cell


title: Hi-C – NPC rep 3

description:
SSEA1-, P-RFP-, X-GFP-, G1
Hi-C_NP_rep_3.hic


title: T-PLL Sample, Patient 18

description: Microarray gene expression data from T-PLL patient 18


title: RNA-seq – ESC rep 1

description:
SSEA1+ P-RFP+, X-GFP+
RNA-seq_ES.txt


title: RNA-seq – iPSC rep 3


title: RNA-seq – iPSC rep 2


title: RNA-seq – iPSC rep 1


title: RNA-seq – ESC rep 3

description:
SSEA1+ P-RFP+, X-GFP+
RNA-seq_ES.txt


title: T-PLL Sample, Patient 10

description: Microarray gene expression data from T-PLL patient 10


title: T-PLL Sample, Patient 12

description: Microarray gene expression data from T-PLL patient 12


title: T-PLL Sample, Patient 15

description: Microarray gene expression data from T-PLL patient 15


title: T-PLL Sample, Patient 9

description: Microarray gene expression data from T-PLL patient 9


title: T-PLL Sample, Patient 13

description: Microarray gene expression data from T-PLL patient 13


title: T-PLL Sample, Patient 8

description: Microarray gene expression data from T-PLL patient 8


title: T-PLL Sample, Patient 5

description: Microarray gene expression data from T-PLL patient 5


title: T-PLL Sample, Patient 7

description: Microarray gene expression data from T-PLL patient 7


title: T-PLL Sample, Patient 14

description: Microarray gene expression data from T-PLL patient 14


title: Vogel_17008_Liver tumor_FAH-/-

type: genomic

city: Ulm

first: André

last: Lechel

address: Albert-Einstein-Allee 23

department: Department of Internal Medicine I

country: Germany

institute: University hospital Ulm

hyb_protocol: Labeled DNA Samples were hybridized for 24 hours at 67°C and 20 rpm rotation speed according to manufacturer´s protocol (Agilent Technologies, Santa Clara, CA, USA).

scan_protocol: Microarray slides were scanned using a G2565CA microarray scanner (Agilent Technologies, Santa Clara, CA, USA).

data_processing: The scanned images were analyzed with the Feature Extraction Software 10.7.3.1 (Agilent).

source_name: liver tumor

label_protocol: 1.5µg of the isolated genomic DNA was digested and labeled by random primer with Cyanine 5-UTP (liver tumor) or Cyanine 3-UTP (reference: liver) using the SureTag Labeling Kit (Agilent Technologies, Santa Clara, CA, USA) according to the manufacturer´s protocol.

growth_protocol: Drinking water was supplemented with 7.5 mg/L NTBC 14 unless otherwise indicated, and mice had access to food (Altromin1324 M, Altromin, Germany) and water ad libitum. Mice were housed in individually ventilated cages and all experiments were performed during the day time of a 12 hour day/night cycle.

extract_protocol: Genomic DNA was isolated from liver and liver tumor tissue according to standard phenol-chloroform extraction protocol.

treatment_protocol: Mice were exposed to a reduced treatment regimen of NTBC (2.5% of the normal dose) for 6 to 12 months

value: FAH-/-

tag: gender

value: male

value: FAH+/+


title: RNA-seq – D10 X-GFP+ rep 4

description:
reprogramming day 10; SSEA1+, P-RFP+, X-GFP+
RNA-seq_TNG.txt


title: RNA-seq – D10 X-GFP+ rep 3


title: Vogel_16014_Liver tumor_FAH-/-


title: RNA-seq – D10 X-GFP+ rep 2


title: T-PLL Sample, Patient 4

description: Microarray gene expression data from T-PLL patient 4


title: Vogel_16012_Liver tumor_FAH-/-


title: T-PLL Sample, Patient 16

description: Microarray gene expression data from T-PLL patient 16


title: Vogel_17012_Liver tumor_FAH-/-


title: input G1E-ER4+E2 [573]

city: University Park

first: Ross

last: Hardison

state: PA

address: 303 Wartik Lab

institute: Pennsylvania State University

data_processing:
Basecalls using bcl2fastq-1.8.4, and parameters --no-eamss --mismatches 1
Mapping to reference genome mm10 canon with Bowtie 1.0.0 using parameters --chunkmbs 1024 -y -n 2 --best -k 1 --maxbts 800 -l 28 -e 80 --sam-nohead --sam
Wiggle and peaks called using MACS with parameters --format BAM --gsize 1870000000 --tsize 36 --bw 120 --mfold 12 --wig --space 1
Filter blacklist regions from peaks, and convert the *peaks.xls file from MACs to broadpeak format (see UCSC Genome Browser for format specs).
Genome_build: mm10 (GRCm38)
Supplementary_files_format_and_content: bigWigs, signal from MACS.
Supplementary_files_format_and_content: broadpeaks, peak calls by MACS.

source_name: G1E-ER4+E2 cells

growth_protocol: G1E-ER4 cells were grown in IMDM media + 15% FBS, kit ligand, and erythropoietin in a standard tissue culture incubator at 37°C with 5% CO2.

extract_protocol:
Lysates were prepared from sonicated nuclei and protein-DNA complexes were isolated with antibody.
ChIP-seq samples were prepared by end repair, 3’ adenylation, and adaptor ligation using Illumina’s TruSeq ChIP-seq Sample Preparation Kit.

treatment_protocol: To induce erythroid maturation, G1E-ER4 cells were treated with 10-8 mol/L β-estradiol for 24 hours (G1E-ER4+E2).

value: ES-derived erythroid progenitor cells

tag: chip antibody

tag: average chromatin size in bp as measured between a 100-500 bp window using the agilent bioanalyzer 7500 chip

tag: subjective quality assessment (pass, low pass, fail)


title: input G1E-ER4+E2 [572]


title: input G1E-ER4+E2 [575]


title: Vogel_16013_Liver tumor_FAH-/-


title: ACHN_das_cabo_3

city: Portland

first: Laura

last: Heiser

state: OR

address: 2730 S Moody Ave

department: Biomedical Engineering

institute: Oregon Health and Science University

data_processing:
Single-end, 100bp reads were trimmed of low-quality bases and adapter sequences using TrimGalore (v0.4.4) a wrapper for cutadapt (v1.10).
Trimmed reads were aligned to the human reference genome (GRCh38) and summarized to gene-level counts using STAR (v2.4.0.1).
Genome_build: hg38
Supplementary_files_format_and_content: tab-separated matrix table of raw, unnormalized read counts for all genes in all samples.

extract_protocol:
Total RNA was extracted from cells using a Qiagen RNeasy Mini kit, and RNA-seq libraries were prepared using an Illumina TruSeq RNA Library Prep kit.
Unstranded polyA RNA-seq libraries were prepared using an Illumina TruSeq RNA Library Prep kit.

tag: cell line

value: ACHN

value: Renal cell carcinoma cells

value: wild type

tag: treatment

tag: cabozantinib concentration

value: 50 nM

tag: dasatinib concentration

value: 10 nM

tag: timepoint


title: ACHN_das_cabo_2

tag: cell type


title: Vogel_16011_Liver tumor_FAH-/-


title: T-PLL Sample, Patient 2

description: Microarray gene expression data from T-PLL patient 2

value: Peripheral Blood Mononuclear Cells


title: T-PLL Sample, Patient 3

description: Microarray gene expression data from T-PLL patient 3


title: RNA-seq – D9 X-GFP+ rep 4

description:
reprogramming day 9; SSEA1+, P-RFP+, X-GFP+
RNA-seq_9NG.txt

value: hybrid 129S1/SvImJ, CAST/EiJ


title: ACHN_das_cabo_1


title: RNA-seq – D9 X-GFP+ rep 3


title: input G1E-ER4+E2 [574]


title: Vogel_16009_Liver tumor_FAH-/-


title: T-PLL Sample, Patient 1

description: Microarray gene expression data from T-PLL patient 1


title: CTCF G1E-ER4+E2 [408]

value: CTCF (Millipore 07-729)

value: Fail


title: 240_Liver tumor_FAH-/- Trp53-/-

data_processing: The scanned images were analyzed with the Feature Extraction Software 12.0.0 (Agilent).

label_protocol: 1µg of the isolated genomic DNA was digested and labeled by random primer with Cyanine 5-UTP (liver tumor) or Cyanine 3-UTP (reference: liver) using the SureTag Labeling Kit (Agilent Technologies, Santa Clara, CA, USA) according to the manufacturer´s protocol.

treatment_protocol: Mice were exposed to a reduced treatment regimen of NTBC (2.5% of the normal dose) for 6 months

value: liver tumor

value: liver


title: 244_Liver tumor_FAH-/- Trp53-/-


title: CTCF G1E-ER4+E2 [377]


title: Vogel_16010_Liver tumor_FAH-/-


title: CTCF G1E-ER4+E2 [374]


title: 252_Liver tumor_FAH-/- Trp53-/-


title: CTCF G1E-ER4+E2 [299]

value: Pass


title: 239_Liver tumor_FAH-/- Trp53-/-


title: RNA-seq – D9 X-GFP+ rep 2

value: induced pluripotent stem cell


title: RNA-seq – D9 X-GFP+ rep 1


title: ACHN_cabo_2

value: cabozantinib

value: 0 nM


title: ACHN_cabo_1


title: ACHN_cabo_3


title: ACHN_das_3

value: dasatinib


title: RNA-seq – D8 X-GFP+ rep 3

description:
reprogramming day 8; SSEA1+, P-RFP+, X-GFP+
RNA-seq_8NG.txt


title: ACHN_das_2


title: RNA-seq – D8 X-GFP+ rep 2


title: RNA-seq – D8 X-GFP+ rep 4


title: RNA-seq – D10 X-GFP+ rep 1


title: ACHN_das_1


title: ACHN_veh_2

value: untreated


title: RNA-seq – D7 X-GFP+ rep 4

description:
reprogramming day 7; SSEA1+, P-RFP+, X-GFP+
RNA-seq_7NG.txt


title: ACHN_veh_3

value: ACHN


title: ACHN_veh_1

value: Renal cell carcinoma cells


title: RNA-seq – D8 X-GFP+ rep 1


title: TPLL_9

description: small non-coding RNA sequencing of PBMC from TPLL patient

data_processing:
After sequencing and de-multiplexing the quality of the data was assessed with fastQC (v 0.11.8).
When the RNA sequence data passed the quality check, fastq files were aligned using STAR aligner (v.2.5.3e) with GRCh38 as reference.
The small non-coding RNA reads were trimmed and aligned with Novalign, using novoalign indexes.
Read counts were obtained using FeatureCounts (v1.6.0) and Homo_sapiens GRCh38.92 as reference and miRBase.annotation20 for miRNA sequence data.
The quality of the alignment and assigned reads were determined with MultiQC.Noise reduction was applied to the count data by removing the very low read counts.
Normalized data was used for visualization and comparison purposes, which include correlation and clustering analysis generating the heatmaps and PCA plots.
For the differential expression analysis, DESeq2 (R package v1.22.2) was used.
Genome_build: GRCh38
Supplementary_files_format_and_content: Tab-delimetd tekst file containing RNA counts per gene

source_name: Peripheral Blood Mononuclear Cells

growth_protocol: Peripheral blood from healthy donors were obtained from buffy coats (Sanquin) and T-PLL patient samples were obtained with approval by the Erasmus MC Medical Ethics Committee (MEC-2016-202). For gene expression profiling, peripheral blood mononuclear cells (PBMC) were first isolated by Ficoll-Paque (density 1.077 g/ml, Pharmacia). CD4+ T cells were labeled with anti-CD3-PerCP-Cy5.5 and anti-CD4-PE-Cy7 and CD8+ T cells were labeled with anti-CD3-PerCP-Cy5.5 and anti-CD8-APC-H7) (BD Biosciences). Naïve T cells (CD27+ and CD45RA+), memory (CD27+ and CD45RO+ and effector (CD27- and CD45RA+) T cells subsets were labeled with, anti-CD45RA-PE, anti-CD27-APC, anti-CD45RO-FITC, anti-CD27-APC and anti-CD45RA-PE antibodies (BD Biosciences)(Supplementary Table 1). Cells fractions were sorted on a FACS Aria II cell sorter (BD Biosciences). When the tumor load of a patient was below 85%, cells were purified using cell sorting on a FACSAria III, based on the CD4 and CD8 expression on T-PLL cells.

extract_protocol:
T-PLL cells were lysed in Trizol (Invitrogen) and total RNA was isolated according to manufacturer’s protocol. Quality and concentration of total RNA of the samples was determined with the RNA 6000 Nano assay on an Aligent 2100 Bioanalyzer (Agilent Technologies).
The small non-coding RNA cDNA libraries were generated with 100 ng total RNA using CleanTag Small RNA Library Preparation Kit (Trilink Biotechnologies) according to the manufacturer’s protocol.

tag: sorted

value: Unsorted

value: T-PLL


title: RNA-seq – D7 X-GFP+ rep 3


title: RNA-seq – D7 X-GFP+ rep 2


title: TPLL_29


title: RNA-seq – D7 X-GFP+ rep 1


title: TPLL_17


title: TPLL_26


title: TPLL_28


title: RNA-seq – D6 X-GFP+ rep 4

description:
reprogramming day 6; SSEA1+, P-RFP+, X-GFP+
RNA-seq_6NG.txt


title: TPLL_4


title: RNA-seq – D6 X-GFP+ rep 3


title: TPLL_25


title: TPLL_3


title: TPLL_2


title: TPLL_24


title: TPLL_5


title: TPLL_23


title: TPLL_16


title: RNA-seq – D6 X-GFP+ rep 2


title: TPLL_10


title: TPLL_1


title: TPLL_22


title: TPLL_18


title: CD8mem28_6

description: small non-coding RNA sequencing of CD8 memory T-cells sorted out of healthy donor PBMCs

value: healthy


title: CD8mem22_6

value: Peripheral Blood Mononuclear Cells


title: TPLL_21


title: RNA-seq – D6 X-GFP~ rep 3

description:
reprogramming day 6; SSEA1+, P-RFP+, X-GFP intermediate
RNA-seq_6NM.txt

value: hybrid 129S1/SvImJ, CAST/EiJ


title: RNA-seq – D6 X-GFP~ rep 2


title: TPLL_20


title: CD4mem28_6

description: small non-coding RNA sequencing of CD4 memory T-cells sorted out of healthy donor PBMCs


title: CD4naive22_6

description: small non-coding RNA sequencing of CD4 naive T-cells sorted out of healthy donor PBMCs


title: CD8naive22_6

description: small non-coding RNA sequencing of CD8 effector T-cells sorted out of healthy donor PBMCs


title: CD4eff15_6 (sRNA)

description: small non-coding RNA sequencing of CD4 effector T-cells sorted out of healthy donor PBMCs


title: TPLL_19


title: RNA-seq – D6 X-GFP~ rep 1


title: RNA-seq – D6 P-RFP+ rep 4

description:
reprogramming day 6; SSEA1+, P-RFP+, X-GFP-
RNA-seq_6NN.txt


title: Pombe_CHX_rep2

city: Solna

first: Vicent

last: Pelechano

address: Nobels väg 16

department: MTC

country: Sweden

institute: ScilifeLab - Karolinska Institutet

description:
Pombe-CHX_2
HT-5Pseq with CHX (0.1 mg/mL)  in S.pombe

data_processing:
library strategy: RNA-Seq (5PSeq)
Base-calling was done using bcl2fastq v2.20.0.422  and for demultiplex, we allowed one mismatch in index1 and one mismatch in index2
For HT-5Pseq data, 3’-sequencing adaptor (-AGATCGGAAGAGCACACGTCTGAACTCCAGTCA) trimming was applied to 5’ends of reads using cutadapt V1.16. The 8-nt random barcodes on the 5’ ends of reads were extracted and added to the header of fastq file as the UMI using UMItools (v0.5.4). Reads were mapped to the reference genome (SGD R64-1-1 for S. cerevisiae genome, ASM294v2.20 for S.pombe genome) separately by star/2.7.0  with the parameter --alignEndsType Extend5pOfRead1 to exclude soft-clipped bases on the 5’ end. To calculate the fraction of rRNA, tRNA, snRNA snoRNA and mRNA in library compositions,the stepwise alignment was performed by corresponding index generated by star/2.7.0. Duplicated 5’ends of read introduced by PCR during library preparation were removed based on random barcodes sequences using UMItools (v0.5.4).
For The distribution of nucleotide position of 5’ends were performed using Fivepseq package , including relative to start, stop codon and codon specific pausing. Specifically, the unique 5’mRNA reads in biological samples were summed up and normalized to reads per million (rpm). Then the relative position of 5’mRNA reads to all codons of all ORF were summed at each position. The metagene plot was showed as the sum value versus the relative distance from respective codon.
Genome_build: Saccharomyces cerevisiae R64-1-1 , Schizosaccharomyces pombe ASM294v2
Supplementary_files_format_and_content: Bedgraph files for UMI collapsed reads for positive and negative strand. Reads were collapsed to the first 5´nucleotide.

organism: Schizosaccharomyces pombe

growth_protocol: S. cerevisiae  grown in YPD at 30°C. S.pombe grown in YES at 30°C

extract_protocol:
Total RNA was isolated by the standard phenol: chloroform method, and DNA was removed by DNase I treatment. The amount of RNA was quantified in a Nanodrop 2000 Spectrophotometer (Thermo Scientific) and RNA integrity was checked by agarose gel electrophoresis.
For HT5Pseq libraries construction, we used 6 μg of DNA-free total RNA. Samples were directly subjected to RNA ligation. The treated RNA samples were incubated with 100 μM RNA/RNA rP5_RND oligo (final 10 μM) 2h at 25°C with 10 Units of T4 RNA ligase 1 (NEB). Ligated RNA was purified with RNAClean XP (Beckman Coulter), according to the manufacturer’s instructions. RNA was reverse transcribed with Superscript II (Life Technologies) and primed with Illumina PE2 compatible oligos containing random hexamers (20 μM) and oligo-dT (0.05 μM). Reverse transcription reaction was incubated for 10 min at 25°C, 50 min at 42°C and heat inactivation for 15 min at 70°C. To deplete RNA in RNA/cDNA hybrid after reverse transcription, we used sodium hydroxide (40 mM) for incubation 20 min at 65°C and then neutralized with Tris-HCl, pH =7.0 (40 mM). For DSN (Duplex-specific nuclease) based rRNA depletion, we used a mixture of probes targeted the 18S rDNA, 25S rDNA and 5.8S rDNA. The probes were designed to occupy the whole ribosomal RNA regions with consecutive 25-30nt long unmodified DNA oligos. The hybridization of probes (2 μM each) with cDNAs were incubated at 68 °C for 2 minutes before adding pre-warmed DSN buffer mix with 1 Units of DSN enzyme (Evrogen). The reaction then performed at 68 °C for 20 minutes. To inactive DSN enzyme, we added 2X DSN stop solution and incubate 10 min at 68 °C. The final PCR amplification was used 2X Phusion High-Fidelity PCR Master Mix with HF Buffer (NEB) and final 0.1 μM of PE1.0 and corresponding multiplex PE2.0_MTX . The program followed this: 30s 98°C; 15 cycles (20s 98°C; 30s 65°C; 30s 72°C); 7min 72°C. Libraries were size selected using 0.7x-0.9x (v/v) AMpure XP beads (Beckman Coulter) to final 200-500 bp and sequenced by NextSeq 500/550 using 60 sequencing cycles for Read 1 and 15 cycles for Read 2.  5PSeq methods was performed as previously described (PMID: 26820793). 6µg of total RNA was used as input. In brief a RNA oligo (rP5_RND) containing an Illumina adaptor and unique molecular identifiers (UMI) was ligated to the intermediates of mRNA co-translation degradation (5’P). Ribosomal RNA was depleted using Ribo-Zero Magnetic Gold Kit (Illumina).Libraries were PCR amplified (15 cycles). Ampure beads size selected libraries with an average length of 451 nt were sent for sequencing (llumina NextSeq 500 instrument).

treatment_protocol: For cycloheximide (CHX) treatment, CHX was added to final 0.1 mg/mL to the medium and incubated for 5 min at 30°C for S. cerevisiae and 10 min for S.pombe. For glucose deprivation, cells were shifted to pre-warmed YP (lacking glucose) and then grown at 30°C for 5 minutes and 15 minutes prior to harvesting. For glucose re-addition, cultures with already 15 min glucose deprivation were shifted to YPD for 15 min at 30°C. For early exponential phase, strains were grown at 30°C from an initial OD600~0.05 to a final OD600 of 0.3. To reach stationary stage, S. cerevisiae strains were grown during 60h in YPD.

tag: strain background

value: h-

tag: parental strain background

value: Schizosaccharomyces pombe h-


title: Pombe_CHX_rep1

description:
Pombe-CHX_1
HT-5Pseq with CHX (0.1 mg/mL)  in S.pombe


title: TPLL_11


title: Pombe_NonDSN_rep2

description:
Pombe-NonDSN_2
HT-5Pseq without DSN treatment in S.pombe


title: Pombe_NonDSN_rep1

description:
Pombe-NonDSN_1
HT-5Pseq without DSN treatment in S.pombe


title: Pombe_DSN_rep2

description:
Pombe-DSN_2
HT-5Pseq with DSN treatment and probes in S.pombe


title: RNA-seq – D6 P-RFP+ rep 2

tag: strain


title: RNA-seq – D6 P-RFP+ rep 3


title: Pombe_DSN_rep1

description:
Pombe-DSN_1
HT-5Pseq with DSN treatment and probes in S.pombe


title: TCRgd_pos_8_6

description: small non-coding RNA sequencing of CD8 naive T-cells sorted out of healthy donor PBMCs

tag: tissue

value: TCRgd


title: TCRgd_pos_4_5

tag: disease state


title: Genomic DNA from primary myoblasts  CP6

first: Andrea

middle: A.

last: Domenighetti

address: 345 East Superior St

institute: Northwestern University & Rehabilitation Institute of Chicago

description:
Genomic DNA from primary myoblasts
From contractured muscle in a child with cerebral palsy

hyb_protocol: A 500 ng DNA sample was used to perform bisulfite conversion followed by Illumina’s protocol for methylation profiling. Bisulphite converted DNA was amplified, fragmented and hybridised to Illumina Infinium Human Methylation850K Beadchip using standard Illumina protocol.

scan_protocol: BeadChips were scanned with an Illumina iScan using standard recommended Illumina scanner setting.

data_processing: Data were analyzed using the Illumina GenomeStudio software to obtain beta- and M-values (an estimate of methylation levels) for each CpG. Values were processed largely following the procedure described in Maksimovic et al. (PMID: 27347385). Briefly, beadChip data were checked for quality by calculating detection p-values using minfi. Quantile normalization was performed on the data to account for sample variation. Low quality probes were filtered from the dataset along with probes with SNPs at the CpG site, probes that bind to multiple locations, and probes on sex chromosomes. After normalization and filtering, a total of 775,687 specific CpG probes with unique beta-values were retained for discovery and annotation of differentially methylated regions (DMRs). DMRcate was used to identify differentially methylated regions, while a motif enrichment analysis using HOMER (Hypergeometric Optimization of Motif EnRichment) was used to annotate them. A DMR was positively identified when it included at least 3 differentially methylated CpGs in close proximity and a log10-ratio above 0.3 (hypermethylation) or below -0.3 (hypomethylation).

label: Cy5 and Cy3

source_name: Hamstring muscle biopsy

label_protocol: Standard Illumina Protocol

growth_protocol: Satellite cell-derived myoblasts were cultured at 37°C in a 5% CO2 atmosphere using a high-serum medium containing Ham’s F10 (Corning) with 20% Fetal Bovine Serum (FBS, from Gibco), 10 mg/ml streptomycin and 100 units/ml penicillin (Gibco) and human recombinant bFGF (5 ng/ml) (BD). Myoblasts were seeded at ~5,000 cells/cm2 and were maintained in the same high-serum medium up to 20-30% of confluence before 5-azacytidine (AZA) treatment. Cells were treated with AZA (5 μM) (Sigma-Aldrich) for 24 hours to induce DNA demethylation, or not treated with AZA as a control. After 24h, the AZA-containing medium was removed, cells were rinsed twice with high-serum medium and then cultured until they reached 70-80% confluence in high-serum medium before DNA extraction.

extract_protocol: Genomic DNA was isolated and purified from 70-80% confluent myoblast cell culture preparations (passage 4). All DNA extractions were completed using a Puregene Cell & Tissue Kit (Qiagen, MD) according to manufacturer’s recommended protocol for cultured cells. During DNA extraction procedures, samples were treated with RNase A solution (10 minutes at 37°C).

value: Male

tag: age

tag: gmfcs

tag: limbs

value: Diplegic


title: TCRgd_pos_28_6


title: Genomic DNA from primary myoblasts  CP2_5AZA

description:
Genomic DNA from primary myoblasts
From contractured muscle in a child with cerebral palsy, treated with 5-azacytidine

treatment_protocol: Cells were treated with AZA (5 μM) (Sigma-Aldrich) for 24 hours to induce DNA demethylation, or not treated with AZA as a control. After 24h, the AZA-containing medium was removed, cells were rinsed twice with high-serum medium and then cultured until they reached 70-80% confluence in high-serum medium before DNA extraction.

tag: gender


title: RNA-seq – D6 X-GFP~ rep 4

value: induced pluripotent stem cell


title: Genomic DNA from primary myoblasts  CP2


title: Genomic DNA from primary myoblasts  TD5

description:
Genomic DNA from primary myoblasts
From typically developing muscle (control)


title: RNA-seq – D6 X-GFP+ rep 1

description:
reprogramming day 6; SSEA1+, P-RFP+, X-GFP+
RNA-seq_6NG.txt


title: Genomic DNA from primary myoblasts  CP5_5AZA

value: Female

value: Quadriplegic


title: CD103_lung_Lcn2KO_4

city: Vienna

first: Sylvia

last: Knapp

address: Währinger Gürtel 18-20

country: Austria

institute: Medical University of Vienna

description:
Low-input RNA-Seq of CD103+ DCs
LCN2_homeostasis_lung.csv
CD103_lung_Lcn2KO_4

data_processing:
Illumina HiSeq Control Software (HCS) HD 3.4.0.38 was used for the data acquisition on the sequencing instrument.
On-instrument base-calling performed by the Illumina Real-Time Analysis (RTA) software 2.7.7.
BCL to BAM file conversion and demultiplexing with custom programmes based on Picard tools 2.18.24 and 2.19.2 (http://broadinstitute.github.io/picard/)
Trimming for adapter sequences with Trimmomatic 0.39 in single-end (SE) mode with ILLUMINACLIP:TruSeq3-SE.fa:2:30:10:1:true SLIDINGWINDOW:4:15 MINLEN:20
Spliced genome alignments to GRCh38 (UCSC Genome Browser hg38) and Ensembl reference transcriptome e96 (April 2019) with STAR 2.5.2b and options suggested be the ENCODE project (--alignIntronMax 1000000 --alignIntronMin 20 --alignMatesGapMax 1000000 --alignSJDBoverhangMin 1 --alignSJoverhangMin 8 --outFilterMismatchNmax 999 --outFilterMismatchNoverReadLmax 0.04 --outFilterMultimapNmax 20 --outFilterType BySJout)
Genome_build: mm10
Supplementary_files_format_and_content: Matrix of read counts (CSV), read counts mapping to genes.

source_name: Murine CD103+ dendritic cells

growth_protocol: Experiments were conducted using specific opportunistic pathogen free (SOPF) age-matched 8- to 12-week-old C57BL/6J WT or Lcn2–/– mice.

extract_protocol:
Lungs were digested using DNAse I and Collagenase I. CD103+ DCs were isolated using FACS.
For each sample, 200 cells were sorted into 96-well plates containing 4 μl cell lysis buffer (nuclease-free H2O [Life Technologies] with 0.2% Triton X-100 [Sigma] and RNase Inhibitor [2 U/µl, Takara/Clonentech]). Cell lysates were stored at -80°C until library preparation according to the Smart-Seq2 protocol (Picelli et al, 2014).
Libraries were constructed using the Nextera XT DNA Sample Preparation Kit with 1ng of cDNA.

tag: organ

value: Lung

tag: condition

value: Steady state

value: 9-11 weeks

tag: genotype


title: Genomic DNA from primary myoblasts  CP1


title: Genomic DNA from primary myoblasts  TD8


title: CD103_lung_Lcn2KO_3

description:
Low-input RNA-Seq of CD103+ DCs
LCN2_homeostasis_lung.csv
CD103_lung_Lcn2KO_3


title: Genomic DNA from primary myoblasts  CP5


title: CD103_lung_WT_4

description:
Low-input RNA-Seq of CD103+ DCs
LCN2_homeostasis_lung.csv
CD103_lung_WT_4

value: WT


title: Genomic DNA from primary myoblasts  CP6_5AZA


title: RNA-seq – D5 P-RFP+ rep 4

description:
reprogramming day 5; SSEA1+, P-RFP+, X-GFP-
RNA-seq_5NN.txt


title: Genomic DNA from primary myoblasts  TD8_5AZA

description:
Genomic DNA from primary myoblasts
From typically developing muscle (control), treated with 5-azacytidine


title: CD103_lung_Lcn2KO_2

description:
Low-input RNA-Seq of CD103+ DCs
LCN2_homeostasis_lung.csv
CD103_lung_Lcn2KO_2


title: TCRgd_pos_22_6


title: CD103_lung_Lcn2KO_1

description:
Low-input RNA-Seq of CD103+ DCs
LCN2_homeostasis_lung.csv
CD103_lung_Lcn2KO_1


title: RNA-seq – D6 P-RFP+ rep 1

description:
reprogramming day 6; SSEA1+, P-RFP+, X-GFP-
RNA-seq_6NN.txt


title: Genomic DNA from primary myoblasts  TD5_5AZA


title: RNA-seq – D5 P-RFP+ rep 3


title: Genomic DNA from primary myoblasts  TD2


title: CD103_lung_WT_1

description:
Low-input RNA-Seq of CD103+ DCs
LCN2_homeostasis_lung.csv
CD103_lung_WT_1


title: Genomic DNA from primary myoblasts  CP1_5AZA


title: Genomic DNA from primary myoblasts  TD1


title: Genomic DNA from primary myoblasts  TD1_5AZA


title: Genomic DNA from primary myoblasts  TD2_AZA


title: RNA-seq – D4 P-RFP+ rep 4

description:
reprogramming day 4; SSEA1+, P-RFP+, X-GFP-
RNA-seq_4NN.txt


title: CD8naive28_6


title: RNA-seq – D5 P-RFP+ rep 2


title: CD103_lung_WT_3

description:
Low-input RNA-Seq of CD103+ DCs
LCN2_homeostasis_lung.csv
CD103_lung_WT_3


title: AMs_lung_Lcn2KO_1

description:
Low-input RNA-Seq of Ams
LCN2_homeostasis_lung.csv
AMs_lung_Lcn2KO_1

source_name: Murine alveolar macrophages

extract_protocol:
Lungs were digested using DNAse I and Collagenase I. AMs were isolated using FACS.
For each sample, 200 cells were sorted into 96-well plates containing 4 μl cell lysis buffer (nuclease-free H2O [Life Technologies] with 0.2% Triton X-100 [Sigma] and RNase Inhibitor [2 U/µl, Takara/Clonentech]). Cell lysates were stored at -80°C until library preparation according to the Smart-Seq2 protocol (Picelli et al, 2014).
Libraries were constructed using the Nextera XT DNA Sample Preparation Kit with 1ng of cDNA.


title: CD103_lung_WT_2

description:
Low-input RNA-Seq of CD103+ DCs
LCN2_homeostasis_lung.csv
CD103_lung_WT_2


title: CD8naive8_6

description: small non-coding RNA sequencing of CD8 naive T-cells sorted out of healthy donor PBMCs


title: RNA-seq – D5 P-RFP+ rep 1


title: AMs_lung_WT_4

description:
Low-input RNA-Seq of Ams
LCN2_homeostasis_lung.csv
AMs_lung_WT_4


title: RNA-seq – D4 P-RFP+ rep 2


title: AMs_lung_WT_3

description:
Low-input RNA-Seq of Ams
LCN2_homeostasis_lung.csv
AMs_lung_WT_3


title: CD8naive4_5


title: AMs_lung_Lcn2KO_3

description:
Low-input RNA-Seq of Ams
LCN2_homeostasis_lung.csv
AMs_lung_Lcn2KO_3


title: CD8mem8_6


title: AMs_lung_Lcn2KO_4

description:
Low-input RNA-Seq of Ams
LCN2_homeostasis_lung.csv
AMs_lung_Lcn2KO_4


title: CD8mem15_6

description: small non-coding RNA sequencing of CD8 memory T-cells sorted out of healthy donor PBMCs


title: TCRgd_pos_15_6

description: small non-coding RNA sequencing of TCRgd positive sorted out of healthy donor PBMCs


title: CD8mem4_5


title: AMs_lung_WT_2

description:
Low-input RNA-Seq of Ams
LCN2_homeostasis_lung.csv
AMs_lung_WT_2


title: AMs_lung_Lcn2KO_2

description:
Low-input RNA-Seq of Ams
LCN2_homeostasis_lung.csv
AMs_lung_Lcn2KO_2


title: AMs_lung_WT_6

description:
Low-input RNA-Seq of Ams
LCN2_homeostasis_lung.csv
AMs_lung_WT_6


title: RNA-seq – D4 P-RFP+ rep 3


title: AMs_lung_WT_5

description:
Low-input RNA-Seq of Ams
LCN2_homeostasis_lung.csv
AMs_lung_WT_5


title: CD103_lung_Lcn2KO_5

description:
Low-input RNA-Seq of CD103+ DCs
LCN2_homeostasis_lung.csv
CD103_lung_Lcn2KO_5


title: AMs_lung_Lcn2KO_5

description:
Low-input RNA-Seq of Ams
LCN2_homeostasis_lung.csv
AMs_lung_Lcn2KO_5


title: CD8naive15_6


title: RNA-seq – D4 P-RFP+ rep 1

description:
reprogramming day 4; SSEA1+, P-RFP+, X-GFP-
RNA-seq_4NN.txt


title: CD103_lymphnode_WT_1

description:
Low-input RNA-Seq of CD103+ DCs
LCN2_infection_mLN.csv
CD103_lymphnode_WT_1

extract_protocol:
Lymph nodes were digested using DNAse I and Liberase TL. CD103+ DCs were isolated using FACS
For each sample, 200 cells were sorted into 96-well plates containing 4 μl cell lysis buffer (nuclease-free H2O [Life Technologies] with 0.2% Triton X-100 [Sigma] and RNase Inhibitor [2 U/µl, Takara/Clonentech]). Cell lysates were stored at -80°C until library preparation according to the Smart-Seq2 protocol (Picelli et al, 2014).
Libraries were constructed using the Nextera XT DNA Sample Preparation Kit with 1ng of cDNA.

value: Mediastinal lymph node

value: OT1-PR/8 Influenza infection

value: 12 weeks


title: RNA-seq – D4 SSEA1+ rep 4

description:
reprogramming day 4; SSEA1+, P-RFP-, X-GFP-
RNA-seq_4SN.txt


title: RNA-seq – D4 SSEA1+ rep 2


title: AMs_lung_WT_1

description:
Low-input RNA-Seq of Ams
LCN2_homeostasis_lung.csv
AMs_lung_WT_1


title: CD8eff4_5

description: small non-coding RNA sequencing of CD8 effector T-cells sorted out of healthy donor PBMCs

value: Peripheral Blood Mononuclear Cells

value: healthy


title: CD103_lymphnode_Lcn2KO_2

description:
Low-input RNA-Seq of CD103+ DCs
LCN2_infection_mLN.csv
CD103_lymphnode_Lcn2KO_2


title: CD103_lymphnode_Lcn2KO_1

description:
Low-input RNA-Seq of CD103+ DCs
LCN2_infection_mLN.csv
CD103_lymphnode_Lcn2KO_1


title: CD8eff28_6


title: RNA-seq – D4 SSEA1+ rep 1

value: hybrid 129S1/SvImJ, CAST/EiJ


title: RNA-seq – NPC rep 4

description:
SSEA1-, P-RFP-, X-GFP-
RNA-seq_NP.txt

value: neural progenitor cell


title: UN_CT_3_ham_S90_L004 reanalysis

city: La Jolla

first: Debashis

last: Sahoo

address: 9500 Gillman Drive

department: Pediatrics

institute: UCSD

data_processing:
RNASeq data was processed using kallisto (version 0.45.0), Mesocricetus auratus genome (MesAur1.0 ). Gene-level TPM values and gene annotations were computed using tximport and biomaRt R package. A custom python script was used to organize the data and log reduced using log2(TPM) if TPM > 1 and TPM - 1 if TPM <= 1.
Genome_build: kallisto index on Mesocricetus_auratus.MesAur1.0.ncrna.fa.gz + Mesocricetus_auratus.MesAur1.0.cdna.all.fa.gz.
Supplementary_files_format_and_content: tab-delimited text files include log2(TPM) if TPM > 1 else TPM - 1 values for each Sample.

organism: Mesocricetus auratus

source_name: Lung

growth_protocol: Hamster lung samples were collected after two different treatment regimens.

extract_protocol:
The lungs were harvested from the hamsters and homogenized in TRI-Reagent. RNA were extracted after infection using the Quick-RNA MicroPrep Kit (Zymo Research, USA) according to the manufacture’s instruction. RNA was converted into cDNA using the qScript™ cDNA SuperMix (Quantabio).
RNA sequencing libraries were generated using the Illumina TruSeq Stranded Total RNA Library Prep Gold with TruSeq Unique Dual Indexes (Illumina, San Diego, CA). Samples were processed following manufacturer’s instructions, except modifying RNA shear time to five minutes. Resulting libraries were multiplexed and sequenced with 100 basepair (bp) Paired End (PE100) to a depth of approximately 25-40 million reads per sample on an Illumina NovaSeq 6000. Samples were demuxltiplexed using bcl2fastq v2.20 Conversion Software (Illumina, San Diego, CA).

treatment_protocol: Hamster were infected with SARS-CoV-2 using a BSL-3 facility.

value: Untreated

tag: infection

value: Uninfected


title: RNA-seq – D4 SSEA1+ rep 3


title: UN_CT_1_ham_S89_L004 reanalysis


title: CD103_lymphnode_WT_2

description:
Low-input RNA-Seq of CD103+ DCs
LCN2_infection_mLN.csv
CD103_lymphnode_WT_2


title: CD8eff15_6


title: CD8eff22_6


title: RNA-seq – NPC rep 3

description:
SSEA1-, P-RFP-, X-GFP-
RNA-seq_NP.txt


title: RNA-seq – NPC rep 2

description:
SSEA1-, P-RFP-, X-GFP-
RNA-seq_NP.txt


title: Namir_7_663_S94

value: Vehicle Treated

value: Infected


title: CD4naive8_6

description: small non-coding RNA sequencing of CD4 naive T-cells sorted out of healthy donor PBMCs


title: Namir_7_666_S96


title: Namir_6_646_S92

value: EIDD-2801 Treated


title: Namir_7_664_S95


title: RNA-seq – NPC rep 1

description:
SSEA1-, P-RFP-, X-GFP-
RNA-seq_NP.txt


title: Namir_6_644_S91


title: CD4naive4_5


title: CD103_lymphnode_Lcn2KO_3

description:
Low-input RNA-Seq of CD103+ DCs
LCN2_infection_mLN.csv
CD103_lymphnode_Lcn2KO_3


title: Namir_6_643_S90


title: CD4naive28_6


title: ATAC-seq – ESC rep 4

description:
SSEA1+ P-RFP+, X-GFP+
ATAC-seq_ES.txt.gz

value: embryonic stem cell


title: ATAC-seq – ESC rep 3


title: CD4naive15_6


title: Namir_6_642_S89


title: ATAC-seq – ESC rep 2


title: CD4mem4_5

description: small non-coding RNA sequencing of CD4 memory T-cells sorted out of healthy donor PBMCs


title: TEA-seq of leukapheresis-purified, 0.01% digitonin perm, FACS neutrophil-depleted PBMCs, method comparison, well 4

description:
X066-MP0C1W6_leukopak_perm-cells_tea_200M_atac_filtered_fragments.tsv.gz
X066-MP0C1W6_leukopak_perm-cells_tea_200M_atac_filtered_metadata.csv.gz
X066-MP0C1W6_leukopak_perm-cells_tea_200M_cellranger-arc_filtered_feature_bc_matrix.h5
X066-MP0C1W6_leukopak_perm-cells_tea_200M_cellranger-arc_per_barcode_metrics.csv.gz
X066-MP0C1W6_leukopak_perm-cells_tea_48M_adt_counts.csv.gz
X066-MP0C1W6
The FASTQs are from human subjects and will be provided via dbGAP
OTHER: TEA-seq

data_processing:
Demultiplexing of raw base call files into FASTQ files was performed using bcl2fastq2 (Illumina v2.20.0.422, parameters: --create-fastq-for-index-reads, --minimum-trimmed-read-length=8, --mask-short-adapter-reads=8, --ignore-missing-positions, -ignore-missing-filter, --ignore-missing-bcls, -r 24 -w 24 -p 80). The --use-bases-mask option was used to adjust the read lengths for each library type as follows: Y28n*,I10,I10n*,Y90n* for Gene Expression, Y50n*,I8n*,Y16,Y50n* for ATAC, and Y28n*,I8n*,n*,Y90n* for ADT.
For analysis of TEA-seq data alone, the full available sequencing depth from each library for each well was used for alignment. To perform comparisons between methodologies, all scRNA-seq and scATAC-seq libraries were downsampled to 200 million FASTQ entries (200M of each sequenced read; ~20,000 reads per expected cell recovered at ~10,000 cell expected recovery), and all ADT libraries were downsampled to 48M reads. RNA and ATAC libraries were aligned using cellranger-arc software (v1.0.0, 10x Genomics) against 10x genomics reference refdata-cellranger-arc-GRCh38-2020-A using default parameters. ADT barcode counting was performed using BarCounter.
genome_build: Ensembl GRCh38-3.0.0
processed_data_files_format_and_content: Fragment TSVs (Tab separated), Single cell CSVs (Commas separated), and HDF5 file (Hierarchical Data Format).

source_name: peripheral blood mononuclear cells

extract_protocol:
Biological specimens were purchased from BioIVT as cryopreserved PBMCs. All sample collections were conducted by BioIVT under IRB-approved protocols, and all donors sign informed consent forms. PBMCs sourced from BioIVT were isolated using leukapheresis. Following isolation, PBMCs were subjected to RBC lysis, washing, and counting. PBMC aliquots were cryopreserved in Cryostor CS10 (StemCell Technologies, 07930) and stored in vapor phase liquid nitrogen.
Cryopreserved PBMCs were thawed, stained for FACS, depleted of neutrophils, dead cells, and debris through FACS, stained with TotalSeq-A antibodies, and permeabilized using 0.01% digitonin. ATAC and Gene Expression libraries were prepared according to the Chromium Next GEM Single Cell Multiome ATAC + Gene Expression User Guide (CG000338 Rev A) with several modifications. To generate a high-quality reference dataset, we generated four 10x Genomics wells of TEA-seq. For each well, transposition was performed by diluting 15,400 permeabilized cells to a final volume of 5 µl in isotonic Tagmentation Buffer (20 mM Tris-HCl pH 7.4, 150 mM NaCl, 3 mM MgCl2, RNase Inhibitor 1U/µl (Lucigen NxGen, F83923-1)). Cells were mixed with 10 µl of a transposition master mix consisting of 7 µl ATAC Buffer B (10x Genomics, 2000193) and 3 µl ATAC Enzyme B (Tn5 transposase; 10x Genomics, 2000265) per reaction. Transposition was performed by incubating the prepared reactions on a C1000 Touch thermal cycler with 96–Deep Well Reaction Module (Bio-Rad, 1851197) at 37°C for 60 minutes, followed by a brief hold at 4°C. A Chromium NextGEM Chip J (10x Genomics, 2000264) was placed in a Chromium Next GEM Secondary Holder (10x Genomics, 3000332) and 50% Glycerol (Teknova, G1798) was dispensed into all unused wells. A master mix composed of Barcoding Reagent Mix (10x Genomics, 2000267), Reducing Agent B (10x Genomics, 2000087), Template Switch Oligo (10x Genomics, 3000228), and Barcoding Enzyme Mix (10x Genomics, 2000266) was then added to each sample well, pipette-mixed, and loaded into row 1 of the chip. Chromium Single Cell Multiome Gel Beads v1.1 (10x Genomics, 2000261) were vortexed for 30 seconds and loaded into row 2 of the chip, along with Partitioning Oil (10x Genomics, 2000190) in row 3. A 10x Gasket (10x Genomics, 3000072) was placed over the chip and attached to the Secondary Holder. The chip was loaded into a Chromium Single Cell Controller instrument (10x Genomics, 120270) for GEM generation. At the completion of the run, GEMs were collected, and reverse transcription and barcoding were performed by incubating GEMs on a C1000 Touch thermal cycler with 96–Deep Well Reaction Module at 37°C for 45 min, 25°C for 30 min, followed by a brief hold at 4°C. Upon completion of the GEM incubation 5 µl of Quenching Agent (10x Genomics, 2000269) was immediately added and mixed with the reaction solution.   GEMs were separated into a biphasic mixture through addition of Recovery Agent (10x Genomics, 220016), the aqueous phase was retained and cleared of barcoding reagents using Dynabead MyOne SILANE (10x Genomics, 2000048) and 2.0x bead:sample ratio SPRIselect reagent (Beckman Coulter, B23318) cleanups, incubating for 10 minutes after each bead addition step.  Barcoded ATAC and cDNA fragments were amplified using a PCR reaction consisting of 45 µl of template, 50 µl of Pre-Amp Mix (10x Genomics, 2000270), 5 µl of Pre-Amp Primers (10x Genomics, 2000271), and 1 µl of ADT-Rev-AMP (0.2 µM Supplementary File 5). Amplification was performed in a C1000 Touch thermal cycler with 96–Deep Well Reaction Module: 72°C for 5 min, 98°C for 3 min, for 7 cycles of: 98°C for 20 sec, 63°C for 30 sec, 72°C for 1 min, with a final extension of 72°C for 1 min. Amplified fragments were purified using a 2.0x bead:sample ratio SPRIselect reagent (Beckman Coulter, B23318) bead clean-up, incubating for 10 minutes after bead addition.  Sequencing ready ATAC libraries were constructed by amplifying barcoded ATAC fragments in a sample indexing PCR consisting of SI-PCR Primer B (10x Genomics, 2000128), Amp Mix (10x Genomics, 2000047) and Sample Index Plate N, Set A (10x Genomics, 3000427) as described in the 10x Multiome User Guide. Amplification was performed in a C1000 Touch thermal cycler with 96–Deep Well Reaction Module: 98°C for 45 sec, then 9 cycles of: 98°C for 20 sec, 67°C for 30 sec, 72°C for 20 sec, with a final extension of 72°C for 1 min. Final ATAC libraries were prepared using a dual-sided 0.6x/1.6x bead:sample SPRIselect reagent (Beckman Coulter, B23318) size-selection clean-up.  cDNA fragments were amplified using a PCR reaction consisting of 35 µl of template, 50 µl of Amp Mix (10x Genomics, 2000047), 15 µl of cDNA Primers (10x Genomics, 2000089), and 1 µl of ADT-Rev-AMP (2 µM Supplementary File 5). Amplification was performed in a C1000 Touch thermal cycler with 96–Deep Well Reaction Module: 98°C for 3 min, then 8 cycles of: 98°C for 15 sec, 63°C for 20 sec, 72°C for 1 min, with a final extension of 72°C for 1 min. cDNA and ADT fragments were separated using a dual-sided SPRIselect reagent (Beckman Coulter, B23318) size-selection clean-up. Large cDNA fragments were retained in an initial 0.6x bead:sample SPRIselect incubation, reactions were incubated on a magnet, and small unbound fragments containing ADTs were transferred to new wells where they were subjected to an additional 2.0x bead:sample SPRIselect cleanup.  ADT fragments were amplified in a 15 cycle indexing PCR, substituting SI-PCR-Oligo primer (10 µM Supplementary File 5) for SI-P5-22. Final ADT libraries were prepared using a dual-sided 1.6x bead:sample SPRIselect reagent (Beckman Coulter, B23318) clean-up.  cDNA Fragmentation, End-Repair, and A-tailing were performed in a joint reaction containing 25% of cDNA sample as described in the 10x Multiome User Guide. Briefly, cDNA was diluted in Buffer EB (Qiagen, 1014609), and combined with master mix of Fragmentation Buffer (10x Genomics, 2000091) and Fragmentation Enzyme (10x Genomics, 2000090). The reaction was incubated in a C1000 Touch thermal cycler with 96–Deep Well Reaction Module: 4°C start, 32°C for 5 min, 65°C for 30 min, followed by a brief hold at 4°C. Reactions were purified using a dual-sided 0.6x/0.8x bead:sample SPRIselect reagent (Beckman Coulter, B23318) size-selection clean-up. Ligation was performed by mixing each sample with a master mix consisting of Ligation Buffer (10x Genomics, 2000092), DNA Ligase (10x Genomics, 220110), and Adapter Oligos (10x Genomics, 2000094), and incubating in a C1000 Touch thermal cycler with 96–Deep Well Reaction Module for 15 minutes at 20°C, followed by a brief hold at 4°C. Ligation reactions were purified using a 0.8x bead:sample SPRIselect reagent (Beckman Coulter, B23318) clean-up.  Sequencing ready Gene Expression libraries were constructed by amplifying cDNA fragments in a sample indexing PCR consisting of Amp Mix (10x Genomics, 2000047) and Dual Index TT Set A (10x Genomics, 3000431) as described in the 10x Multiome User Guide. Amplification was performed in a C1000 Touch thermal cycler with 96–Deep Well Reaction Module: 98°C for 45 sec, for 14 cycles of: 98°C for 20 sec, 54°C for 30 sec, 72°C for 20 sec, with a final extension of 72°C for 1 min. Final Gene Expression libraries were prepared using a dual-sided 0.6x/0.8x bead:sample SPRIselect reagent (Beckman Coulter, B23318) size-selection clean-up.

value: BioIVT

value: leukapheresis

value: permeabilized cells

value: 0.01% digitonin

value: FACS

value: Dead Cell/Debris/Neutrophil Depletion

value: TEA-seq


title: CD4mem8_6

value: Peripheral Blood Mononuclear Cells


title: Namir_7_662_S93


title: CD4mem22_6

value: healthy


title: TEA-seq of leukapheresis-purified, 0.01% digitonin perm, FACS neutrophil-depleted PBMCs, method comparison, well 3

description:
X066-MP0C1W5_leukopak_perm-cells_tea_200M_atac_filtered_fragments.tsv.gz
X066-MP0C1W5_leukopak_perm-cells_tea_200M_atac_filtered_metadata.csv.gz
X066-MP0C1W5_leukopak_perm-cells_tea_200M_cellranger-arc_filtered_feature_bc_matrix.h5
X066-MP0C1W5_leukopak_perm-cells_tea_200M_cellranger-arc_per_barcode_metrics.csv.gz
X066-MP0C1W5_leukopak_perm-cells_tea_48M_adt_counts.csv.gz
X066-MP0C1W5
The FASTQs are from human subjects and will be provided via dbGAP
OTHER: TEA-seq


title: ATAC-seq – iPSC rep 4

description:
reprogramming day 15/16; SSEA1+, P-RFP+, X-GFP+
ATAC-seq_iPS.txt.gz

value: hybrid 129S1/SvImJ, CAST/EiJ

value: induced pluripotent stem cell