title: TEA-seq of leukapheresis-purified, 0.01% digitonin perm, FACS neutrophil-depleted PBMCs, method comparison, well 2

description:
X066-MP0C1W4_leukopak_perm-cells_tea_200M_atac_filtered_fragments.tsv.gz
X066-MP0C1W4_leukopak_perm-cells_tea_200M_atac_filtered_metadata.csv.gz
X066-MP0C1W4_leukopak_perm-cells_tea_200M_cellranger-arc_filtered_feature_bc_matrix.h5
X066-MP0C1W4_leukopak_perm-cells_tea_200M_cellranger-arc_per_barcode_metrics.csv.gz
X066-MP0C1W4_leukopak_perm-cells_tea_48M_adt_counts.csv.gz
X066-MP0C1W4
The FASTQs are from human subjects and will be provided via dbGAP
OTHER: TEA-seq


title: TEA-seq of leukapheresis-purified, 0.01% digitonin perm, FACS neutrophil-depleted PBMCs, method comparison, well 1

status: Public on Apr 02 2021

type: SRA

city: Seattle

first: Allen Institute

last: For Immunology

state: Washington

address: 615 Westlake Ave N, Seattle, WA

country: USA

institute: Allen Institute

description:
X066-MP0C1W3_leukopak_perm-cells_tea_200M_atac_filtered_fragments.tsv.gz
X066-MP0C1W3_leukopak_perm-cells_tea_200M_atac_filtered_metadata.csv.gz
X066-MP0C1W3_leukopak_perm-cells_tea_200M_cellranger-arc_filtered_feature_bc_matrix.h5
X066-MP0C1W3_leukopak_perm-cells_tea_200M_cellranger-arc_per_barcode_metrics.csv.gz
X066-MP0C1W3_leukopak_perm-cells_tea_48M_adt_counts.csv.gz
X066-MP0C1W3
The FASTQs are from human subjects and will be provided via dbGAP
OTHER: TEA-seq

library_source: transcriptomic

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).

molecule: total RNA

organism: Homo sapiens

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: Multiome RNA/ATAC of leukapheresis-purified, isolated nuclei, FACS neutrophil-depleted PBMCs, method comparison

description:
X066-MP0C1W1_leukopak_nuclei_multiome_200M_atac_filtered_fragments.tsv.gz
X066-MP0C1W1_leukopak_nuclei_multiome_200M_atac_filtered_metadata.csv.gz
X066-MP0C1W1_leukopak_nuclei_multiome_200M_cellranger-arc_filtered_feature_bc_matrix.h5
X066-MP0C1W1_leukopak_nuclei_multiome_200M_cellranger-arc_per_barcode_metrics.csv.gz
X066-MP0C1W1
The FASTQs are from human subjects and will be provided via dbGAP
OTHER: 10x Genomics Multiome ATAC + RNA-Seq

library_source: genomic

data_processing:
Demultiplexing of raw base call files into FASTQ files was performed using bcl2fastq2 (Illumina v2.20.0.422
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). 10x Multiome 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.
--minimum-trimmed-read-length=8
--mask-short-adapter-reads=8
--ignore-missing-positions
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).

molecule: genomic DNA

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.
Unstained FACS sorted PBMCs were used as input to Nuclei and permeabilized cell Multiome ATAC plus Gene Expression library preparation. Nuclei were isolated according to 10x Genomics Demonstrated Protocol CG000365 Rev A exactly as recommended for cryopreserved PBMCs. Cells were permeabilized using 0.01% digitonin. Libraries were prepared according to the 10x Genomics Chromium Next GEM Single Cell Multiome ATAC plus Gene Expression user guide CG000338 Rev A.

value: nuclei

value: 10x Multiome


title: Multiome RNA/ATAC of leukapheresis-purified, 0.01% digitonin perm, FACS neutrophil-depleted PBMCs, method comparison

description:
X066-MP0C1W2_leukopak_perm-cells_multiome_200M_atac_filtered_fragments.tsv.gz
X066-MP0C1W2_leukopak_perm-cells_multiome_200M_atac_filtered_metadata.csv.gz
X066-MP0C1W2_leukopak_perm-cells_multiome_200M_cellranger-arc_filtered_feature_bc_matrix.h5
X066-MP0C1W2_leukopak_perm-cells_multiome_200M_cellranger-arc_per_barcode_metrics.csv.gz
X066-MP0C1W2
The FASTQs are from human subjects and will be provided via dbGAP
OTHER: 10x Genomics Multiome ATAC + RNA-Seq

data_processing:
Demultiplexing of raw base call files into FASTQ files was performed using bcl2fastq2 (Illumina v2.20.0.422
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). 10x Multiome 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.
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).


title: CD4eff28_6 (sRNA)

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

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: tissue

value: Peripheral Blood Mononuclear Cells

tag: sorted

tag: disease state

value: healthy


title: ATAC-seq – iPSC rep 1

city: Scarborough

first: Guillaume

middle: J

last: Filion

state: Ontario

address: 1265 Military Trail

department: Biological Sciences

country: Canada

institute: UTSC

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

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

organism: Mus musculus

source_name: induced pluripotent 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.

tag: strain

value: hybrid 129S1/SvImJ, CAST/EiJ

tag: cell type

value: induced pluripotent stem cell


title: PBMC_A.HD2_Adult healthy donor; BCR

city: New Haven

first: Carrie

middle: L

last: Lucas

address: 300 Cedar St., S625D

institute: Yale University

data_processing:
The sequencing data was processed using CellRanger v3.1.0.
Genome_build: GRCh38
Supplementary_files_format_and_content: CellRanger processed .mtx and .tsv files representing barcodes, features, and matrix for each dataset

source_name: PBMC

growth_protocol: Fresh or thawed PBMCs

extract_protocol:
Fresh or thawed PBMCs were centrifuged at 400 g for 8 minutes at RT, and the supernatant was removed without disrupting the cell pellet. The pellet was resuspended in 1X PBS with 0.04% BSA, and cells were filtered with a 30 μM cell strainer. Cellular concentration was adjusted to 1,000 cells/μl based on the cell count and cells were immediately loaded onto the 10x Chromium Next GEM Chip G, according to the manufacturer's user guide (Chromium Next GEM SingleCell V(D)J Reagent Kits v1.1). We aimed to obtain a yield of ~10,000 cells per lane. For CITE-seq staining, lyophilized Total-seq C human cocktail (BioLegend) (Table S2) was resuspended with 35 μL of 2% FBS in PBS vortexed for 10 sec and incubated for 5 min at RT. To pellet the aggregated antibodies, rehydrated antibody cocktail was centrifuged at 20,000g for 10 min just before adding to the cells. PBMCs were resuspended with wash buffer at the concentration of 10-20 x 106 cells/ml, and 0.5 x 106 cells were used for further staining. Cells were incubated on ice for 10 mins with 5ul of Human Fc block and 5 μL of TrueStain Monocyte Blocker (Biolegend). Next, 10-20 μL (0.1-0.2 x 106 cells) were aliquoted into a new tube and incubated on ice for 30 mins with 5 μL of Total-seq C antibody cocktail prepared as above. Cells were washed twice with wash buffer and third wash was with 2% FBS in PBS, then resuspended in 1X PBS with 0.04% BSA at 1,000 cells/ul and loaded onto the 10x Chromium Chip G, as described above.
cDNA libraries for gene expression, CITE-seq, and TCR/BCR sequencing were generated according to manufacturer’s instructions (Chromium Next GEM SingleCell V(D)J Reagent Kits v1.1). Each library was then sequenced on an Illumina Novaseq 6000 platform.

tag: disease subtype

value: Periperal blood mononuclear cells


title: PBMC_A.HD3_Adult healthy donor; BCR


title: PBMC_A.HD1_Adult healthy donor; BCR


title: ATAC-seq – D10 X-GFP+ rep 4

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


title: CD103_lung_WT_5

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_WT_5

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.

molecule: polyA RNA

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

tag: age

value: 9-11 weeks

tag: genotype

value: WT


title: PBMC_C.HD6_Pediatric healthy donor; BCR


title: ATAC-seq – ESC rep 1

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

source_name: embryonic stem cell

value: embryonic stem cell


title: CD4eff22_6 (sRNA)


title: ATAC-seq – D10 X-GFP+ rep 3


title: PBMC_C.HD5_Pediatric healthy donor; BCR


title: PBMC_C.HD4_Pediatric healthy donor; BCR


title: TPLL2

description: mRNA Seq data from TPLL samples

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.
Read counts were obtained using FeatureCounts (v1.6.0) and Homo_sapiens GRCh38.92 as reference
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 raw RNA counts per gene

source_name: T-PLL

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).
cDNA libraries were generated with 1 ng total RNA using the Smart-Seq V4 Ultra low input RNA kit (Takara-bio) in combination with the Truseq nano DNA library prep kit (Illumina).


title: CD4mem15_6

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


title: ATAC-seq – iPSC rep 2


title: PBMC_C.HD3_Pediatric healthy donor; BCR


title: CD4eff8_6 (sRNA)


title: TPLL4

description: mRNA Seq data from TPLL samples


title: PBMC_C.HD1_Pediatric healthy donor; BCR


title: PBMC_P4.2_MIS-C Moderate; Recovered; BCR

value: Multisystem inflammatory syndrome in children (MIS-C)

value: Moderate; Recovered


title: PBMC_C.HD2_Pediatric healthy donor; BCR


title: PBMC_P3.2_MIS-C Severe; Recovered; BCR

value: Severe; Recovered


title: ATAC-seq – D10 X-GFP+ rep 2


title: TPLL3

description: mRNA Seq data from TPLL samples


title: TPLL25

description: mRNA Seq data from TPLL samples


title: PBMC_P7.1_MIS-C Severe; BCR

value: Severe


title: ATAC-seq – D10 X-GFP+ rep 1


title: PBMC_P6.1_MIS-C Severe; BCR


title: CD8eff8_6

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


title: PBMC_P5.1_MIS-C Moderate; BCR

value: Moderate


title: ATAC-seq – D9 X-GFP+ rep 4

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


title: ATAC-seq – D9 X-GFP+ rep 3


title: PBMC_P4.1_MIS-C Moderate; BCR


title: PBMC_P3.1_MIS-C Severe; BCR


title: ATAC-seq – D9 X-GFP+ rep 2


title: TPLL23

description: mRNA Seq data from TPLL samples


title: TPLL24

description: mRNA Seq data from TPLL samples


title: PBMC_P1.1_MIS-C Severe; BCR


title: PBMC_P2.1_MIS-C Severe; BCR


title: TPLL22

description: mRNA Seq data from TPLL samples


title: PBMC_A.HD3_Adult healthy donor; TCR


title: ATAC-seq – D8 X-GFP+ rep 4

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


title: PBMC_A.HD2_Adult healthy donor; TCR


title: TPLL1

description: mRNA Seq data from TPLL samples


title: TPLL17

description: mRNA Seq data from TPLL samples


title: PBMC_C.HD6_Pediatric healthy donor; TCR


title: PBMC_A.HD1_Adult healthy donor; TCR


title: PBMC_C.HD5_Pediatric healthy donor; TCR


title: ATAC-seq – D8 X-GFP+ rep 3


title: TPLL16

description: mRNA Seq data from TPLL samples


title: PBMC_C.HD3_Pediatric healthy donor; TCR


title: TPLL10

description: mRNA Seq data from TPLL samples


title: PBMC_C.HD2_Pediatric healthy donor; TCR


title: ATAC-seq – D8 X-GFP+ rep 2


title: PBMC_C.HD1_Pediatric healthy donor; TCR


title: ATAC-seq – D8 X-GFP+ rep 1


title: PBMC_P4.2_MIS-C Moderate; Recovered; TCR


title: CD4eff8_6

description: mRNA Seq from sorted CD4 effector T-cell fraction from buffy coats

value: T-PLL


title: ATAC-seq – D9 X-GFP+ rep 1


title: PBMC_P3.2_MIS-C Severe; Recovered; TCR


title: PBMC_P6.1_MIS-C Severe; TCR


title: CD4eff28_6

source_name: sorted CD4 effector T-cell fraction from buffy coats


title: ATAC-seq – D7 X-GFP+ rep 4

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


title: PBMC_P7.1_MIS-C Severe; TCR


title: ATAC-seq – D7 X-GFP+ rep 3


title: ATAC-seq – D7 X-GFP+ rep 2


title: PBMC_P5.1_MIS-C Moderate; TCR


title: PBMC_P4.1_MIS-C Moderate; TCR


title: ATAC-seq – D7 X-GFP+ rep 1


title: PBMC_P3.1_MIS-C Severe; TCR


title: PBMC_C.HD4_Pediatric healthy donor; TCR


title: PBMC_P2.1_MIS-C Severe; TCR


title: PBMC_A.HD3_Adult healthy donor; GEX and ADT


title: ATAC-seq – D6 X-GFP+ rep 4

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


title: PBMC_P1.1_MIS-C Severe; TCR


title: PBMC_A.HD2_Adult healthy donor; GEX and ADT


title: ATAC-seq – D6 X-GFP+ rep 3


title: PBMC_A.HD1_Adult healthy donor; GEX and ADT


title: PBMC_C.HD5_Pediatric healthy donor; GEX


title: embryonic skin epidermis_E13.5_Sas-4_ mutant_3

city: Cologne

first: Hisham

last: Bazzi

state: NRW

address: Joseph-Stelzmann-Straße 26

department: CECAD & Dermatology

country: Germany

institute: University Hospital Cologne

description:
EXPORT_FPKM_RUN2448_KS_E13point5.xlsx
79703_105_3ctrl

data_processing:
For files beginning with K002000098:  Basecalling: RTA v2.7.6; Demultiplexing und Adapter-Trimming: bcl2fastq2 v2.17.1.14; QC: FastQC v0.11.5
For files beginning with K002000183: Basecalling: RTA v2.7.7; Demultiplexing und Adapter-Trimming: bcl2fastq2 v2.18.0.12; QC: FastQC v0.11.5
For all files: QuickNGS version 1.2.7 with Ensemble version 87 were used for splicing-aware alignment, reference-giuded transcriptome assembly and analysis of differential gene expression.
Genome_build: mm10
Supplementary_files_format_and_content: spreadsheet files include FPKM values for each sample

source_name: embryonic skin epidermis

growth_protocol: Newborn pups were sacrificed at P0 or pregnant females were dissected to harvest embryos at E13.5 for further skin preparation.

extract_protocol:
embryonic skin from Sas-4 mutant and control mice was dissected at E13.5 and incubated with trypsin/pancreatin soultion for 5 min at RT and then for 30 min on ice to separate epidermal sheets from the underlying dermal tissue. Epidermal samples were frozen until further RNA isolation.
RNA isolation was performed by following the manufacturer's instructions of the Quiagen RNeasy Mini Kit. Total RNA samples were used for RNA-Seq analysis.
RNA libraries were prepared for sequencing using standard Illumina protocols

value: embryonic day E13.5

value: K14-Cre tg/wt; Sas-4 fl/fl


title: ATAC-seq – D6 X-GFP+ rep 1


title: embryonic skin epidermis_E13.5_Sas-4_ mutant_2

description:
EXPORT_FPKM_RUN2448_KS_E13point5.xlsx
79705_105_4ctrl


title: PBMC_C.HD6_Pediatric healthy donor; GEX

value: healthy


title: PBMC_C.HD4_Pediatric healthy donor; GEX


title: CD4eff22_6

value: Peripheral Blood Mononuclear Cells


title: ATAC-seq – D6 X-GFP~ rep 4

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

value: hybrid 129S1/SvImJ, CAST/EiJ

value: induced pluripotent stem cell


title: PBMC_C.HD2_Pediatric healthy donor; GEX

value: Periperal blood mononuclear cells


title: PBMC_C.HD1_Pediatric healthy donor; GEX


title: PBMC_C.HD3_Pediatric healthy donor; GEX


title: embryonic skin epidermis_E13.5_Sas-4_ control_4

description:
EXPORT_FPKM_RUN2448_KS_E13point5.xlsx
79693_67_2mut

value: K14-Cre wt/wt; Sas-4 fl/wt


title: CD4eff15_6


title: ATAC-seq – D6 X-GFP~ rep 3


title: embryonic skin epidermis_E13.5_Sas-4_ control_3

description:
EXPORT_FPKM_RUN2448_KS_E13point5.xlsx
79695_67_5mut


title: embryonic skin epidermis_E13.5_Sas-4_ mutant_4

description:
EXPORT_FPKM_RUN2448_KS_E13point5.xlsx
79701_105_2ctrl


title: ATAC-seq – D6 X-GFP~ rep 2


title: PBMC_P7.1_MIS-C Severe; GEX


title: PBMC_P6.1_MIS-C Severe; GEX


title: ATAC-seq – D6 X-GFP~ rep 1


title: PBMC_P5.1_MIS-C Moderate; GEX


title: ATAC-seq – D6 X-GFP+ rep 2


title: ATAC-seq – D6 P-RFP+ rep 4

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


title: embryonic skin epidermis_E13.5_Sas-4_ control_2

description:
EXPORT_FPKM_RUN2448_KS_E13point5.xlsx
79697_105_6mut


title: PBMC_P3.2_MIS-C Severe; Recovered; GEX


title: USC L2_LNC

city: Shenzhen

first: Yuqi

last: Wu

state: Guangdong

address: Xueyuan Road 1098

country: China

institute: Shenzhen University

data_processing:
The paired-end sequencing mode of the Illumina Hiseq 3000 sequencing platform was used for high-throughput sequencing of multiple samples, and the Fastp software was used to remove the adapter sequence fragments and low-quality fragments. BWA(v0.7.10 http://bio-bwa.sourceforge.net/) software was used to compare the preprocessed data with the rRNA sequence database.
Use STAR(2.5.3a https://github.com/alexdobin/STAR) software to compare the preprocessed sequence with the reference genome sequence of the sequenced species, and use RSEQC(v2.6.4 https://sourceforge.net/projects/rseqc/) to count the data.
From Reads comparison to reference genome results, StringTie(v1.3.1c http://ccb.jhu.edu/software/stringtie/ )software is used to assemble the transcripts in the sample. According to the sequence coverage depth information of the connecting region between Exon region and Exon, construct the most likely mRNA structure and estimate its expression level.
According to the definition of lncRNA, a series of screenings were performed on the transcripts assembled by StringTie(v1.3.1c http://ccb.jhu.edu/software/stringtie/ ). Known long-chain non-coding transcript information comes from the Ensembl database(ftp://ftp.ensembl.org/pub/release-91/fasta/rattus_norvegicus/Rattus_norvegicus.Rnor_6.0.91.chr.gtf.gz).
For the previous STAR(2.5.3a https://github.com/alexdobin/STAR) comparison results, CIRCexplorer2(2.2.6 https://github.com/YangLab/CIRCexplorer2) software was used to predict the front and back positions of the circular RNA.
The expression of transcripts is calculated using FPKM (FPKM-Fragments Per Kilobase of transcript per Million fragments mapped).
Genome_build: ftp://ftp.ensembl.org/pub/release-91/fasta/rattus_norvegicus/dna/
Supplementary_files_format_and_content: gene_sample_FPKM.txt  (tab-delimited text file includes FPKM values for each Sample)

organism: Rattus norvegicus

source_name: Bladder tissue

extract_protocol:
RNA degradation and contamination was monitored on 1% agarose gels. RNA purity was checked using the NanoPhotometer® spectrophotometer (IMPLEN, CA, USA). RNA concentration was measured using Qubit® RNA Assay Kit in Qubit® 2.0 Flurometer (Life Technologies, CA, USA). RNA integrity was assessed using the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies, CA, USA). A total amount of 1 μg RNA per sample was used as input material for the RNA sample preparations. Firstly, ribosomal RNA was removed by Epicentre Ribo-zero™ rRNA Removal Kit (Epicentre, USA), and rRNA free residue was cleaned up by ethanol precipitation. Subsequently, sequencing libraries were generated using the rRNA-depleted RNA by NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer’s recommendations. Briefly, fragmentation was carried out using divalent cations under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer（5X）. First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase（RNaseH-）. Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. In the reaction buffer, dNTPs with dTTP were replaced by dUTP. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3’ ends of DNA fragments, NEBNext Adaptor with hairpin loop structure were ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 150~200 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). Then 3 μl USER Enzyme (NEB，USA) was used with size-selected, adaptor-ligated cDNA at 37°C for 15 min followed by 5 min at 95°C before PCR. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. At last, products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system. The clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS (Illumia) according to the manufacturer’s instructions. After cluster generation, the libraries were sequenced on an Illumina Hiseq 3000 platform and 150 bp paired-end reads were generated.
RNA libraries were prepared for sequencing using standard Illumina protocols

value: Sprague-Dawley

tag: treatment

value: human USC

tag: human donor id

value: L

value: Bladder


title: primary mouse epidermal keratinocytes_P0_Sas-4_ mutant_4

description:
EXPORT_FPKM_RUN2424_KS_P0.xlsx
55530_76mut

source_name: primary mouse skin keratinocytes

extract_protocol:
For epidermal keratinocyte isolation, back skin of P0 animals was dissected and incubated overnight at 4°C with dispase II to remove the dermis from the epidermis. Then, epidermal sheets were treated with TrypLE solution to dissociate the keratinocytes. Samples were frozen until further RNA isolation.
RNA isolation was performed by following the manufacturer's instructions of the Quiagen RNeasy Mini Kit. Total RNA samples were used for RNA-Seq analysis.
RNA libraries were prepared for sequencing using standard Illumina protocols

value: postnatal day P0


title: PBMC_P4.1_MIS-C Moderate; GEX

value: Multisystem inflammatory syndrome in children (MIS-C)


title: USC Z1_LNC

value: Z


title: ATAC-seq – D6 P-RFP+ rep 3


title: primary mouse epidermal keratinocytes_P0_Sas-4_ mutant_3

description:
EXPORT_FPKM_RUN2424_KS_P0.xlsx
55528_69mut


title: embryonic skin epidermis_E13.5_Sas-4_ mutant_1

description:
EXPORT_FPKM_RUN2448_KS_E13point5.xlsx
79707_105_5ctrl


title: PBMC_P2.1_MIS-C Severe; GEX and ADT

value: Severe


title: PBMC_P3.1_MIS-C Severe; GEX


title: PBMC_P4.2_MIS-C Moderate; Recovered; GEX

value: Moderate; Recovered


title: ATAC-seq – D6 P-RFP+ rep 1


title: primary mouse epidermal keratinocytes_P0_Sas-4_ mutant_2

description:
EXPORT_FPKM_RUN2424_KS_P0.xlsx
55526_68mut


title: Model 1_LNC

value: Model


title: USC Z2_LNC


title: PBMC_P1.1_MIS-C Severe; GEX and ADT


title: primary mouse epidermal keratinocytes_P0_Sas-4_ mutant_1

description:
EXPORT_FPKM_RUN2424_KS_P0.xlsx
55524_63mut

value: K14-Cre tg/wt; Sas-4 fl/fl


title: ATAC-seq – D6 P-RFP+ rep 2


title: primary mouse epidermal keratinocytes_P0_Sas-4_ control_4

description:
EXPORT_FPKM_RUN2424_KS_P0.xlsx
55538_77ctrl


title: embryonic skin epidermis_E13.5_Sas-4_ control_1

description:
EXPORT_FPKM_RUN2448_KS_E13point5.xlsx
79699_105_8mut

value: embryonic day E13.5


title: Ctrl 2_LNC

value: Ctrl


title: ATAC-seq – iPSC rep 3

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

value: hybrid 129S1/SvImJ, CAST/EiJ


title: primary mouse epidermal keratinocytes_P0_Sas-4_ control_3

description:
EXPORT_FPKM_RUN2424_KS_P0.xlsx
55536_71ctrl

value: K14-Cre tg/wt; Sas-4 fl/wt


title: ATAC-seq – D5 P-RFP+ rep 3

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

value: induced pluripotent stem cell


title: ICICLE-seq of leukapheresis-purified, 0.01% digitonin permeabilized PBMCs, well 3

description:
X044-AP0C1W3_leukopak_perm-cells_icicle_fulldepth_filtered-fragments.tsv.gz
X044-AP0C1W3_leukopak_perm-cells_icicle_fulldepth_filtered-metadata.csv.gz
X044-AP0C1W3_leukopak_perm-cells_icicle_fulldepth_20kb-window-matrix.h5
X044-AP0C1W3
The FASTQs are from human subjects and will be provided via dbGAP
OTHER: ICICLE-seq

data_processing:
Demultiplexing of raw base call files into FASTQ files was performed using bcl2fastq2 (Illumina v2.20.0.422).
Read 2 was trimmed of adapter sequences, low quality bases and reads, and polyA tailing using fastp (S. Chen et al., 2018) v0.21.0 (parameters: --adapter_sequence=CTGTCTCTTATACACATCT --cut_tail --trim_poly_x).
Trimmed read 2 sequences were aligned to the GRCh38 (hg38) reference genome (Illumina iGenomes, https://support.illumina.com/sequencing/sequencing_software/igenome.html) using Bowtie 2 (Langmead and Salzberg, 2012; v2.3.0, parameters: --local, --sensitive, --no-unal, --phred33).
Aligned reads in SAM format were filtered by alignment score (greater than or equal to 30) then tagged with cell barcode and UMI sequence and quality scores using custom python code (python3 v3.7.3). Barcode sequences were compared against the 10x Genomics v3 3’ GEX barcode whitelist (3M-february-2018.txt.gz). Sequences not included in the whitelist were corrected to a valid whitelist barcode by allowing a single base mismatch (Hamming distance of 1). Sequences with more than one possible match were corrected at the position with the lowest sequencing quality score. Reads with barcodes that could not be corrected were excluded from further analysis. Filtered and tagged SAM files were converted to sorted, indexed BAM files using GATK (McKenna et al., 2010; Broad Institute v4.1.4.0). Genomic coordinates were converted to BED format using bedtools (Quinlan and Hall, 2010; v2.26.0). Custom python code was used to collapse aligned fragments into a list of fragments with unique cell barcode and genomic coordinate combinations. These fragments were then written as a fragments.tsv.gz file in the format: chr, start position, end position, cell barcode, UMI count, and strand (+/-).
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).

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.
PBMCs were depleted of neutrophils, dead cells, and debris through FACS. 2×106 sorted PBMCs were 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 DPBS without calcium and magnesium (Corning 21-031-CM) supplemented with 2% w/v BSA (Sigma-Aldrich A2934). 10 µl TruStain FcX (BioLegend, 422302) was added and cells were incubated on ice for 10 minutes. A panel of 46 barcoded oligo-conjugated antibodies (BioLegend TotalSeq-A) including a mouse IgG1? isotype negative control (Supplementary File 6) was added and incubated on ice for 30 minutes. Cells were washed three times in 4 mL of DPBS plus 2% BSA to remove unbound antibodies and used as input into cell permeabilization with 0.01% digitonin.  Transposition was performed by aliquoting 20,000 permeabilized cells in TD1 buffer (Illumina, 15027866), bringing the volume up to 9 µl in TD1 buffer, and mixing with 6 µl of Poly-T overhang Tn5 complexes. Reactions were incubated on a C1000 Touch thermal cycler with 96-Deep Well Reaction Module (Bio-Rad, 1851197) at 37°C for 120 minutes, followed by a brief hold at 4°C. Cell barcodes were then added to ATAC and antibody derived tags (ADTs) via GEM generation using 10x Genomics 3’ RNA beads and subsequent amplification. Briefly, a Chromium Next GEM Chip G (10x Genomics, 2000177) was placed in a Chromium Next GEM Secondary Holder (10x Genomics, 3000332) and 50% Glycerol (Teknova, G1798) was dispensed into all unused wells. A barcoding master mix was prepared which consisted of NEBNext Ultra II Q5 Master Mix (New England Biolabs, M0544), Reducing Agent B (10x Genomics, 2000087), F BC Primer (0.2 µM Supplementary File 5), and ADT-Rev-AMP (0.2 µM Supplementary File 5). The master mix was added to each 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 (10x Genomics, 120270) for GEM generation. At the completion of the run, GEMs were collected and amplification was performed on a C1000 Touch thermal cycler with 96-Deep Well Reaction Module: 72°C for 5 min, 98°C for 30 sec, 12 cycles of: 98°C for 10 sec, 42°C for 30 sec and 65°C for 30 sec, followed by a final extension of 65°C for 1 min.  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 (10x Genomics, 2000048) beads. Next, a dual-sided 0.6x/2.0x bead:sample SPRIselect reagent (Beckman Coulter, B23318) size-selection clean-up was performed to remove large DNA fragments and unused primers. Libraries were split into two reactions in a 3:1 ATAC:ADT ratio and amplified separately using different indexed P7 primers. ATAC fragments were amplified in a 100 µl reaction consisting of Buffer EB (Qiagen, 1014609), Amp Mix (10x Genomics, 2000047), SI-P5-22 primer (20 µM Supplementary File 5), and Chromium i7 Multiplex Kit N Set A (10x Genomics, 3000262). ATAC PCR was performed in a C1000 Touch thermal cycler with 96-Deep Well Reaction Module: 98°C for 45 sec, 7 cycles of: 98°C for 20 sec, 54°C for 30 sec, 72°C for 20 sec, followed by a final extension of 72°C for 1 min. ADT fragments were amplified in a 100 µl reaction consisting of Buffer EB (Qiagen, 1014609), KAPA HiFi HotStart ReadyMix (KAPA Biosystems, KM2602), SI-P5-22 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, 15 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 1.2x bead:sample ratio for ADT libraries and a dual-sided size-selection of 0.4x/1.2x bead:sample ratio for ATAC libraries.  Final libraries were quantified using qPCR (KAPA Biosystems Library Quantification Kit for Illumina, KK4844) on a CFX96 Touch Real-Time PCR Detection System (Bio-Rad, 1855195). Library quality and average fragment size were assessed using a Bioanalyzer (Agilent, G2939A) High Sensitivity DNA chip (Agilent, 5067-4626). Libraries were sequenced on the Illumina NovaSeq platform with the following read lengths: 28 bp read 1 (Cell barcode and UMI), 8 bp i7 index, 100 bp read 2 (ATAC-seq sequence or ADT barcode). A Truseq read 1 primer (0.3 µM Supplementary File 5) was included as a Custom Read 1 primer to mitigate the risk of off-target priming of the standard Illumina Nextera read 1 primer on the partial Nextera R1N sequence included in the mosaic end portion of the Poly-T Tn5 insertion.

value: BioIVT

value: leukapheresis

value: permeabilized cells

value: 0.01% digitonin

value: ICICLE-seq


title: Ctrl 1_LNC

value: Sprague-Dawley

value: Bladder


title: ATAC-seq – D5 P-RFP+ rep 4


title: ICICLE-seq of leukapheresis-purified, 0.01% digitonin permeabilized PBMCs, well 2

description:
X044-AP0C1W2_leukopak_perm-cells_icicle_fulldepth_filtered-fragments.tsv.gz
X044-AP0C1W2_leukopak_perm-cells_icicle_fulldepth_filtered-metadata.csv.gz
X044-AP0C1W2_leukopak_perm-cells_icicle_fulldepth_20kb-window-matrix.h5
X044-AP0C1W2
The FASTQs are from human subjects and will be provided via dbGAP
OTHER: ICICLE-seq


title: USC Z2_SMALL

data_processing:
Quality control: Raw data (raw reads) of fastq format were firstly processed through custom perl and python scripts. In this step, clean datas(clean reads) were obtained by removing reads containing ploy-N, with 5’ adapter contaminants, without 3’ adapter or the insert tag, containing ploy A or T or G or C and low quality reads from raw data. At the same time, Q20, Q30, and GC-content of the raw datas were calculated. Then, chose a certain range of length from clean reads to do all the downstream analyses.
Reads mapping to the reference sequence: The small RNA tags were mapped to reference sequence by Bowtie (Langmead et al., 2009) without mismatch to analyze their expression and distribution on the reference.
Known miRNA alignment: Mapped small RNA tags were used to looking for known miRNA. miRBase20.0 was used as reference, modified software mirdeep2(Friedlander et al., 2011) and srna-tools-cli were used to obtain the potential miRNA and draw the secondary structures. Custom scripts were used to obtain the miRNA counts as well as base bias on the first position of identified miRNA with certain length and on each position of all identified miRNA respectively.
Remove tags from these sources: To remove tags originating from protein-coding genes, repeat sequences, rRNA, tRNA, snRNA, and snoRNA, small RNA tags were mapped to RepeatMasker, Rfam database or those types of datas from the specified species itself.
Novel miRNA prediction: The characteristics of hairpin structure of miRNA precursor can be used to predict novel miRNA. The available software miREvo (Wen et al., 2012) and mirdeep2 (Friedlander et al., 2011) were integrated to predict novel miRNA through exploring the secondary structure, the Dicer cleavage site and the minimum free energy of the small RNA tags unannotated in the former steps. At the same time, custom scripts were used to obtain the identified miRNA counts as well as base bias on the first position with certain length and on each position of all identified miRNA respectively.
Small RNA annotation summary: Summarizing all alignments and annotations obtained before. In the alignment and annotation before, some small RNA tags may be mapped to more than one category. To make every unique small RNA mapped to only one annotation, we follow the following priority rule: known miRNA > rRNA > tRNA > snRNA > snoRNA > repeat > gene > NAT-siRNA > gene > novel miRNA > ta-siRNA. The total rRNA proportion was used a marker as sample quality indicator. Usually it should be less than 60% in plant samples and 40% in animal samples as high quality.
miRNA editing analysis: Position 2~8 of a mature miRNA were called seed region which were highly conserved. The target of a miRNA might be different with the changing of nucleotides in this region. In our analysis pipeline, miRNA which might have base edit could be detected by aligning all the sRNA tags to mature miRNA, allowing one mismatch.
miRNA family analysis: Exploring the occurrence of miRNA families identified from the samples in other species. In our analysis pipeline, known miRNA used miFam.dat (http://www.mirbase.org/ftp.shtml) to look for families; novel miRNA precursor was submitted to Rfam (http://rfam.sanger.ac.uk/search/) to look for Rfam families.
Quantification of miRNA: miRNA expression levels were estimated by TPM (transcript per million) through the following criteria (Zhou et al., 2010): Normalization formula: Normalized expression = mapped readcount/Total reads*1000000
Genome_build: ftp://ftp.ensembl.org/pub/release-91/fasta/rattus_norvegicus/dna/
Supplementary_files_format_and_content: miRNA_all_miRNA_exp.txt  (tab-delimited text file includes TPM values for each Sample)

extract_protocol:
RNA degradation and contamination was monitored on 1% agarose gels. RNA purity was checked using the NanoPhotometer® spectrophotometer (IMPLEN, CA, USA) . RNA concentration was measured using Qubit® RNA Assay Kit in Qubit® 2.0 Flurometer (Life Technologies, CA, USA). RNA integrity was assessed using the RNA Nano 6000 Assay Kit of the Agilent Bioanalyzer 2100 system (Agilent Technologies, CA, USA). A total amount of 1 μg total RNA per sample was used as input material for the small RNA library. Sequencing libraries were generated using NEBNext® Multiplex Small RNA Library Prep Set for Illumina® (NEB, USA.) following manufacturer’s recommendations and index codes were added to attribute sequences to each sample. Briefly, NEB 3' SR Adaptor was directly, and specifically ligated to 3' end of miRNA, siRNA and piRNA. After the 3' ligation reaction, the SR RT Primer hybridized to the excess of 3' SR Adaptor (that remained free after the 3' ligation reaction) and transformed the single-stranded DNA adaptor into a double-stranded DNA molecule. This step is important to prevent adaptor-dimer formation, besides, dsDNAs are not substrates for ligation mediated by T4 RNA Ligase 1 and therefore do not ligate to the 5´ SR Adaptor in the subsequent ligation step. 5´ends adapter was ligated to 5´ends of miRNAs, siRNA and piRNA. Then first strand cDNA was synthesized using M-MuLV Reverse Transcriptase (RNase H–). PCR amplification was performed using LongAmp Taq 2X Master Mix, SR Primer for illumina and index (X) primer. PCR products were purified on a 8% polyacrylamide gel (100V, 80 min). DNA fragments corresponding to 140~160 bp (the length of small noncoding RNA plus the 3' and 5' adaptors) were recovered and dissolved in 8 μL elution buffer. At last, library quality was assessed on the Agilent Bioanalyzer 2100 system using DNA High Sensitivity Chips. The clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq SR Cluster Kit v3-cBot-HS (Illumia) according to the manufacturer’s instructions. After cluster generation, the library preparations were sequenced on an Illumina Hiseq 3000 platform and 50bp single-end reads were generated.
RNA libraries were prepared for sequencing using standard Illumina protocols

value: human USC

value: Z


title: primary mouse epidermal keratinocytes_P0_Sas-4_ control_1

description:
EXPORT_FPKM_RUN2424_KS_P0.xlsx
55532_64ctrl

value: postnatal day P0

value: K14-Cre wt/wt; Sas-4 fl/wt


title: ATAC-seq – D5 P-RFP+ rep 1


title: ATAC-seq – D5 P-RFP+ rep 2


title: USC Z1_SMALL


title: ICICLE-seq of leukapheresis-purified, 0.01% digitonin permeabilized PBMCs, well 1

description:
X044-AP0C1W1_leukopak_perm-cells_icicle_fulldepth_filtered-fragments.tsv.gz
X044-AP0C1W1_leukopak_perm-cells_icicle_fulldepth_filtered-metadata.csv.gz
X044-AP0C1W1_leukopak_perm-cells_icicle_fulldepth_20kb-window-matrix.h5
X044-AP0C1W1
The FASTQs are from human subjects and will be provided via dbGAP
OTHER: ICICLE-seq


title: Model 2_LNC

value: Model


title: Model 2_SMALL


title: Model 1_SMALL


title: ATAC-seq – D4 P-RFP+ rep 3

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


title: scATAC-seq of ficoll-purified, 0.01% digitonin permeabilized, FACS neutrophil-depleted PBMCs from donor RG1131

description:
X041-AP0C1W6_ficoll_perm-cells_neudep-facs_ds200M_filtered-fragments.tsv.gz
X041-AP0C1W6_ficoll_perm-cells_neudep-facs_ds200M_filtered-metadata.csv.gz
X041-AP0C1W6_ficoll_perm-cells_neudep-facs_ds200M_20kb-window-matrix.h5
X041-AP0C1W6
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: ficoll

value: FACS

value: Dead Cell/Debris/Neutrophil Depletion

value: 10x scATAC-seq


title: ATAC-seq – D4 P-RFP+ rep 4

value: hybrid 129S1/SvImJ, CAST/EiJ

value: induced pluripotent stem cell


title: primary mouse epidermal keratinocytes_P0_Sas-4_ control_2

description:
EXPORT_FPKM_RUN2424_KS_P0.xlsx
55534_70ctrl

value: K14-Cre tg/wt; Sas-4 fl/wt


title: ATAC-seq – D4 P-RFP+ rep 2


title: Agonist (C26) treated  2

city: New York

first: Bassem

middle: D

last: Khalil

state: NY

address: 1 Gustave L. Levy Pl

institute: Icahn School of Medicine at Mount Siani

data_processing:
Quality control was performed using FastQC (v0.11.8).
Trim Galore! (version 0.6.5) was used to trim the adapter sequences with a quality threshold of 20. The human genome reference used was GRCh38.p13 and GENCODE release 36.
Alignment was performed using STAR aligner (v2.7.5b)
Gene level read counts were obtained by using Salmon (v1.2.1) for all libraries. All samples passed the quality control requirements with > 60% of reads uniquely mapping (>20M uniquely mapped reads for each library) using STAR.
Differential expression analysis was performed using the gene level read counts and the DESeq2 (v1.28.1) R package
Genome_build: GRCh38.p13
Supplementary_files_format_and_content: CSV file of normalized gene counts from all replicates or differential gene expression

source_name: T-HEp3 HNSCC_treatment

growth_protocol: T-HEp3 GFP cells were grown on chicken embryo chorioallantoic membrane (CAM)

extract_protocol:
RNA was extracted from fresh frozen cell pellets using Qiagen RNeasy Plus Universal mini kit following manufacturer’s instructions (Qiagen, Hilden, Germany). RNA samples received were quantified using Qubit 2.0 Fluorometer (Life Technologies, Carlsbad, CA, USA) and RNA integrity was checked using Agilent TapeStation 4200 (Agilent Technologies, Palo Alto, CA, USA).
RNA sequencing libraries were prepared using the NEBNext Ultra RNA Library Prep Kit for Illumina following manufacturer’s instructions (NEB, Ipswich, MA, USA). Briefly, mRNAs were first enriched with Oligo(dT) beads. Enriched mRNAs were fragmented for 15 minutes at 94°C. First strand and second strand cDNAs were subsequently synthesized. cDNA fragments were end repaired and adenylated at 3’ ends, and universal adapters were ligated to cDNA fragments, followed by index addition and library enrichment by limited-cycle PCR. The sequencing libraries were validated on the Agilent TapeStation (Agilent Technologies, Palo Alto, CA, USA), and quantified by using Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA) as well as by quantitative PCR (KAPA Biosystems, Wilmington, MA, USA).
The sequencing libraries were clustered on 1 lane of a flowcell. After clustering, the flowcell was loaded on the Illumina HiSeq instrument (4000 or equivalent) according to manufacturer’s instructions. The samples were sequenced using a 2x150bp Paired End (PE) configuration. Image analysis and base calling were conducted by the HiSeq Control Software (HCS). Raw sequence data generated from Illumina HiSeq was converted into fastq files and de-multiplexed using Illumina's bcl2fastq 2.17 software. One mismatch was allowed for index sequence identification.

treatment_protocol: T-HEp3-GFP cells were pretreated with DMSO or 0.5 µM C26 (agonist) for 6 days then inoculated on CAM and treated every 24 hours.

value: T-HEp3 HNSCC cells

value: Agonist (C26)


title: Agonist (C26) treated  3


title: USC L2_SMALL

value: L


title: scATAC-seq of ficoll-purified, 0.01% digitonin permeabilized, unsorted PBMCs from donor RG1131

description:
X041-AP0C1W4_ficoll_perm-cells_no-facs_ds200M_fragments.tsv.gz
X041-AP0C1W4_ficoll_perm-cells_no-facs_ds200M_filtered-metadata.csv.gz
X041-AP0C1W4_ficoll_perm-cells_no-facs_ds200M_20kb-window-matrix.h5
X041-AP0C1W4
The FASTQs are from human subjects and will be provided via dbGAP

value: BioIVT

value: permeabilized cells

value: 0.01% digitonin


title: ATAC-seq – D4 P-RFP+ rep 1


title: DMSO treated  3

source_name: T-HEp3 HNSCC_control

value: DMSO


title: USC L1_SMALL

value: Sprague-Dawley

value: human USC

value: Bladder


title: scATAC-seq of ficoll-purified, 1x 10xNIB isolated nuclei, FACS neutrophil-depleted PBMCs from donor RG1131

description:
X041-AP0C1W3_ficoll_nuclei_neudep-facs_ds200M_filtered-fragments.tsv.gz
X041-AP0C1W3_ficoll_nuclei_neudep-facs_ds200M_filtered-metadata.csv.gz
X041-AP0C1W3_ficoll_nuclei_neudep-facs_ds200M_20kb-window-matrix.h5
X041-AP0C1W3
The FASTQs are from human subjects and will be provided via dbGAP

value: ficoll

value: nuclei


title: ATAC-seq – D4 SSEA1+ rep 4

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


title: scATAC-seq of ficoll-purified, 1x 10xNIB isolated nuclei, FACS dead cell/debris-depleted PBMCs from donor RG1131

description:
X041-AP0C1W2_ficoll_nuclei_dead-facs_ds200M_filtered-fragments.tsv.gz
X041-AP0C1W2_ficoll_nuclei_dead-facs_ds200M_filtered-metadata.csv.gz
X041-AP0C1W2_ficoll_nuclei_dead-facs_ds200M_20kb-window-matrix.h5
X041-AP0C1W2
The FASTQs are from human subjects and will be provided via dbGAP

value: FACS

value: Dead Cell/Debris Depletion


title: DMSO treated  2


title: Ctrl 2_SMALL

value: Ctrl


title: ATAC-seq – D4 SSEA1+ rep 3

value: hybrid 129S1/SvImJ, CAST/EiJ

value: induced pluripotent stem cell


title: DMSO treated  1

value: T-HEp3 HNSCC cells


title: Ctrl 1_SMALL


title: scATAC-seq of leukapheresis-purified, 0.01% digitonin permeabilized, magnetic bead anti-CD15-depleted PBMCs

description:
X032-AP0C1W4_leukopak_perm-cells_neudep-bead_ds125M_filtered-fragments.tsv.gz
X032-AP0C1W4_leukopak_perm-cells_neudep-bead_ds125M_filtered-metadata.csv.gz
X032-AP0C1W4_leukopak_perm-cells_neudep-bead_ds125M_20kb-window-matrix.h5
X032-AP0C1W4
The FASTQs are from human subjects and will be provided via dbGAP

value: leukapheresis

value: Magnetic Bead

value: anti-CD15 Depletion

value: 10x scATAC-seq


title: scATAC-seq of ficoll-purified, 1x 10xNIB isolated nuclei, unsorted PBMCs from donor RG1131

description:
X041-AP0C1W1_ficoll_nuclei_no-facs_ds200M_filtered-fragments.tsv.gz
X041-AP0C1W1_ficoll_nuclei_no-facs_ds200M_filtered-metadata.csv.gz
X041-AP0C1W1_ficoll_nuclei_no-facs_ds200M_20kb-window-matrix.h5
X041-AP0C1W1
The FASTQs are from human subjects and will be provided via dbGAP

value: BioIVT


title: HUDEP2_L_C

city: Nicosia

first: Carsten

middle: Werner

last: Lederer

address: 6 Iroon Avenue, Ayios Dometios

department: Molecular Genetics Thalassaemia Department

country: Cyprus

institute: The Cyprus Institute of Neurology and Genetics

data_processing:
Zebra Call (base calling software developed for BGISEQ-500) was used for basecalling
Raw data were filtered to remove low-quality reads, adaptors and other contaminants, and then clean reads were mapped to the reference genome and to other ncRNA databases such as miRBase and Rfam 12.0 by using Bowtie 2 and CMsearch programs.
MiRDeep2 was used to predict novel miRNAs by exploring the characteristic hairpin structure of pre-miRNAs, and Piano was used to predict piRNAs
The small RNA expression level was calculated by using Transcripts Per Million (TPM). DE analysis between sample groups was performed using DEGseq for replicate samples (Wang L, Wang. X (2020). DEGseq: Identify Differentially Expressed Genes from RNA-seq data).
Genome_build: hg18
Supplementary_files_format_and_content: Excel files include raw counts and TPM for each sample

source_name: Umbilical cord blood

growth_protocol:
hCD34+ cells were isolated from PB of healthy individuals by magnetic-activated cell sorting using hCD34 Microbead Kit, human (Miltenyi), after mononuclear cell isolation using Lymphoprep density gradient medium (Axis-Shield), and according to a published protocol (Protocol C). Isolated cells were then expanded in StemSpan SFEM II (Stem Cell Technologies) supplemented with 1 µM Dexamethasone (Sigma-Aldrich), 2 IU/mL EPO (Binocrit 4000 IU/0.4 mL, Sandoz GmbH), 1% StemSpan CC100 (Stem Cell Technologies) and 1× Penicillin-Streptomycin (Thermo Fisher Scientific) at concentrations below 0.5×106 cells/mL. Medium was changed twice a week.
For the induction of differentiation, hCD34+ cells were cultured in Minimum Essential Medium Eagle (Stem Cell Technologies) supplemented with 30% Defined Fetal Bovine Serum (Hyclone), 105 M 2-Mercaptoethanol (Sigma-Aldrich), 10 IU/mL EPO (Binocrit 4000 IU/0.4 mL, Sandoz GmbH) 10 ng/mL hSCF (PeproTech) and 1× Penicillin-Streptomycin (Thermo Fisher Scientific) at a concentration of 0.5–1×106/mL.
HUDEP cells were expanded in StemSpan SFEM II (Stem Cell Technologies) supplemented with 1 µM Dexamethasone (Sigma-Aldrich), 5 μg/mL Doxycycline (Clontech Laboratories), 100 ng/mL Recombinant Human Stem Cell Factor (hSCF) (PeproTech), 3 IU/mL Epoetin alpha (EPO) (Binocrit 4000 IU/0.4 mL, Sandoz GmbH), and 2× Penicillin-Streptomycin (Thermo Fisher Scientific) at concentrations below 0.5×106 cells/mL. Culture medium was changed twice a week and doxycycline was replenished every other day to retain expression of the HPV16-E6/E7 immortalisation system.
To induce erythroid differentiation, cells were cultured in a three-phase erythroid differentiation culture system previously described. Briefly, cells were cultured in Iscove’s Modified Dulbecco’s Medium (Thermo Fisher Scientific) containing 5% Human Serum from plasma male AB (Sigma-Aldrich), 2× Penicillin-Streptomycin (Thermo Fisher Scientific), 1% L-Glutamine (Thermo Fisher Scientific), 200 µg/mL Holo-Transferrin (Sigma-Aldrich), 2 IU/mL Heparin (Sigma-Aldrich), 10 ng/mL Insulin (Sigma-Aldrich), and 3 IU/mL EPO (Binocrit 4000 IU/0.4 mL, Sandoz GmbH). In phase I (days 1–4), culture medium was supplemented with 100 ng/mL hSCF (PeproTech). In phase II (days 5–7), hSCF was withdrawn from culture. In phase III (days 8 and beyond), doxycycline was also removed from culture medium. Cells in differentiation were maintained at 1×106 cells/mL.

extract_protocol:
Total RNA was harvested using TRIzol reagent (Invitrogen). After RNA extraction, small RNAs were size-selected (18-30 nucleotides) using 15% urea-polyacrylamide gel electrophoresis (PAGE).
18-30 bp short-insert library
BGISEQ-500, standard output 40 Million reads per sample, 50bp single-end sequencing reads

value: Umbilical cord blood

tag: stage of erythroid differentation

value: Late (_L)

tag: days in erythroid differentiation culture medium

value: Cell line HUDEP-2


title: Agonist (C26) treated  1

value: Agonist (C26)


title: scATAC-seq of ficoll-purified, 0.01% digitonin permeabilized, FACS dead cell/debris-depleted PBMCs from donor RG1131

description:
X041-AP0C1W5_ficoll_perm-cells_dead-facs_ds200M_filtered-fragments.tsv.gz
X041-AP0C1W5_ficoll_perm-cells_dead-facs_ds200M_filtered-metadata.csv.gz
X041-AP0C1W5_ficoll_perm-cells_dead-facs_ds200M_20kb-window-matrix.h5
X041-AP0C1W5
The FASTQs are from human subjects and will be provided via dbGAP

value: ficoll

value: permeabilized cells

value: 0.01% digitonin


title: ATAC-seq – D4 SSEA1+ rep 2


title: DMSO rep3

city: Harbin

first: Guangyue

last: Li

state: heilongjang

address: No.26 Hexing Road Xiangfang District

department: College of Life Science

institute: Northeast Forestry University

data_processing:
Basecalls performed using Bcl2fastq (v2.17.1.14)
Cutadapt (v1.9.1) was used to get the "clean reads"
Mapping clean reads to hg19 whole genome using HISAT
Fragments Per Kilobase Million (FPKM) was calculated based on the HTSeq (v0.6.1)
Genome_build: UCSC Hg19
Supplementary_files_format_and_content: Matrix table with raw gene counts for every gene and every sample

source_name: Liver

growth_protocol: According to previous description,the cells were cultured in the media as suggested by the ATCC (Manassas, VA, USA), supplied with 10% fetal bovine serum and 1% penicillin-streptomycin, and maintained at 37°C in an atmosphere of 5% CO2. Ellagic acid (EA) used in treating cells had purity of over 95% (Cat# E2250, Sigma-Aldrich) and was dissolved in dimethyl sulfoxide (DMSO) as a stock solution.

extract_protocol: Total RNA of each sample was quantified and qualified by Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA), NanoDrop (Thermo Fisher Scientific Inc.) and 1% agrose gel. 1 μg total RNA with RIN value above 7 was used for following library preparation. Next generation sequencing library preparations were constructed according to the manufacturer’s protocol (NEBNext® Ultra™ RNA Library Prep Kit for Illumina®). Then libraries with different indices were multiplexed and loaded on an Illumina HiSeq instrument according to manufacturer’s instructions (Illumina, San Diego, CA, USA). Sequencing was carried out using a 2x150bp paired-end (PE) configuration.

treatment_protocol: Total RNA was extracted using the TRIzol reagent from the cells after being cultured for 24 h in a medium containing 50 μM EA or an equal volume of DMSO as a control.

value: Liver

tag: cell line

value: Control


title: ATAC-seq – D4 SSEA1+ rep 1

value: hybrid 129S1/SvImJ, CAST/EiJ


title: DMSO rep1


title: DMSO rep2


title: ATAC-seq – NPC rep 4

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

source_name: neural progenitor cell

value: neural progenitor cell


title: HUDEP2_L_A


title: EA rep3

value: Ellagic acid


title: HUDEP2_L_B


title: ATAC-seq – NPC rep 3


title: ATAC-seq – NPC rep 2


title: scATAC-seq of ficoll-purified, 0.01% digitonin permeabilized, undepleted PBMCs

description:
X032-AP0C1W1_ficoll_perm-cells_no-bead_ds125M_filtered-fragments.tsv.gz
X032-AP0C1W1_ficoll_perm-cells_no-bead_ds125M_filtered-metadata.csv.gz
X032-AP0C1W1_ficoll_perm-cells_no-bead_ds125M_20kb-window-matrix.h5
X032-AP0C1W1
The FASTQs are from human subjects and will be provided via dbGAP


title: scATAC-seq of ficoll-purified, 0.01% digitonin permeabilized, magnetic bead anti-CD15-depleted PBMCs

description:
X032-AP0C1W2_ficoll_perm-cells_neudep-bead_ds125M_filtered-fragments.tsv.gz
X032-AP0C1W2_ficoll_perm-cells_neudep-bead_ds125M_filtered-metadata.csv.gz
X032-AP0C1W2_ficoll_perm-cells_neudep-bead_ds125M_20kb-window-matrix.h5
X032-AP0C1W2
The FASTQs are from human subjects and will be provided via dbGAP


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

description:
X027-AP0C1W3_ficoll_perm-cells_neudep-facs_ds125M_filtered-fragments.tsv.gz
X027-AP0C1W3_ficoll_perm-cells_neudep-facs_ds125M_filtered-metadata.csv.gz
X027-AP0C1W3_ficoll_perm-cells_neudep-facs_ds125M_20kb-window-matrix.h5
X027-AP0C1W3
The FASTQs are from human subjects and will be provided via dbGAP

value: FACS

value: Dead Cell/Debris/Neutrophil Depletion


title: scATAC-seq of ficoll-purified, 0.01% digitonin permeabilized, unsorted PBMCs BRH1291132

description:
X027-AP0C1W1_ficoll_perm-cells_no-facs_ds125M_filtered-fragments.tsv.gz
X027-AP0C1W1_ficoll_perm-cells_no-facs_ds125M_filtered-metadata.csv.gz
X027-AP0C1W1_ficoll_perm-cells_no-facs_ds125M_20kb-window-matrix.h5
X027-AP0C1W1
The FASTQs are from human subjects and will be provided via dbGAP


title: EA rep1


title: HUDEP2_E_B

value: Early (_E)


title: HUDEP2_E_C


title: HUDEP2_E_A


title: ATAC-seq – NPC rep 1


title: scATAC-seq of ficoll-purified, 0.2% digitonin permeabilized, unsorted PBMCs

description:
X025-AP0C1W4_ficoll_perm-cells_0.20dig_ds125M_filtered-fragments.tsv.gz
X025-AP0C1W4_ficoll_perm-cells_0.20dig_ds125M_filtered-metadata.csv.gz
X025-AP0C1W4_ficoll_perm-cells_0.20dig_ds125M_20kb-window-matrix.h5
X025-AP0C1W4
The FASTQs are from human subjects and will be provided via dbGAP

value: BloodWorks NW

value: 0.20% digitonin

value: 10x scATAC-seq


title: scATAC-seq of leukapheresis-purified, 0.01% digitonin permeabilized, undepleted PBMCs

description:
X032-AP0C1W3_leukopak_perm-cells_no-bead_ds125M_filtered-fragments.tsv.gz
X032-AP0C1W3_leukopak_perm-cells_no-bead_ds125M_filtered-metadata.csv.gz
X032-AP0C1W3_leukopak_perm-cells_no-bead_ds125M_20kb-window-matrix.h5
X032-AP0C1W3
The FASTQs are from human subjects and will be provided via dbGAP

value: BioIVT

value: leukapheresis

value: Magnetic Bead


title: Patient Samples (mixed samples), scRNA-seq

city: Singapore

first: Hong Kai

last: Lee

address: 8 Biomedical Grove

department: Singapore Immunology Network

country: Singapore

institute: Agency for Science, Technology and Research

description:
Pooled Data (11 samples):
GLY020V1
GLY020V2
GLY020V4
GLY023V1
GLY023V2
GLY023V4
GLY032V1
GLY032V2
GLY032V4
GLY054(HD1)
GLY056(HD2)

data_processing:
Raw sequencing data (BCL files) were demultiplexed and coverted to fastq files using the CellRanger v3.0.2 mkfastq pipline (10x Genomics) and bcl2fastq2 v 2.20.0 (Illumina)
Data quality control, data filtering, cell barcode processing, UMI compression, 5' transcript alignment, and single-cell gene counting was performed using the CellRanger v3.0.2 count pipeline (10x Genomics)
Genome_build: GRCh38-3.0.0
Supplementary_files_format_and_content: Matrix table with raw gene counts for every gene and every sample
Supplementary_files_format_and_content: CellRanger output files (barcodes.tsv, features.tsv, matrix.mtx)

source_name: Peripheral blood mononuclear cells

extract_protocol:
After removing dead cells, monocytes were isolated with the EasySep Human CD14 Positive Selection Kit II. Isolated monocytes (10 million/ml) were then labelled with TotalSeqTM-A antibodies (Biolegend, 1µg) for 30 minutes at 4oC. Cells were washed thrice with BSA (1%) in 1X PBS at 4oC. Two rounds of 10X runs with cell hashing were performed.
Expression libraries were prepared using the 10X Genomics Chromium Single Cell 3′ Library & Gel Bead Kit v2 and i7 Multiplex Kit according to the manufacturer’s protocol (10X Genomics). Hash Tag Oligo libraries were prepared according to CITE-seq and Cell Hashing Protocol from New York Genome Center Technology Innovation Lab.
The libraries were subjected to an indexed paired-end sequencing run of 2x151 cycles on Illumina HiSeq 4000 and/or NovaSeq 6000 at a sequencing depth of 50,000 reads/cell for 3’ gene expression libraries and 2,000 reads/cell for Hash Tag Oligo libraries.

treatment_protocol: Peripheral blood mononuclear cells  were isolated from the blood of healthy controls using Ficoll. CD14+ monocytes were isolated using EasysepTM Human CD14 Positive Selection Kits. Isolated monocytes were rested for 1 hour before downstream analysis. The purity of isolated monocytes were checked using flow cytometry expressions of CD45 and CD14.

tag: infection

value: mixed samples (non-demultiplexed) representing Dengue virus-infected patients and healthy controls

value: Monocytes


title: scATAC-seq of ficoll-purified, 0.1% digitonin permeabilized, unsorted PBMCs

description:
X025-AP0C1W3_ficoll_perm-cells_0.10dig_ds125M_filtered-fragments.tsv.gz
X025-AP0C1W3_ficoll_perm-cells_0.10dig_ds125M_filtered-metadata.csv.gz
X025-AP0C1W3_ficoll_perm-cells_0.10dig_ds125M_20kb-window-matrix.h5
X025-AP0C1W3
The FASTQs are from human subjects and will be provided via dbGAP

source_name: peripheral blood mononuclear cells

value: ficoll

value: permeabilized cells

value: 0.10% digitonin


title: SAS-R EVs

type: RNA

city: Kamakura

first: Satoshi

last: Kondo

state: Kanagawa

address: 10-1 tebiro 6-chome

country: Japan

institute: Toray Industries,Inc.

description: exo-SAS-R'

hyb_protocol: Hybridized for 16 h at 32 C with rotary shake (250 rpm). Hybridization buffer and washing protocol was followed by the protocol supplied by TORAY Industries, Inc..

scan_protocol: 3D-Gene Scanner ((Toray Industries Inc., Tokyo, Japan) was used for scanning. Images were quantified using Extraction(Toray Industries Inc., Tokyo, Japan).

data_processing: The raw data of each spot was normalized by substitution with a mean intensity of the background signal determined by all blank spots’ signal intensities of 95% confidence intervals.

source_name: cell EVs

label_protocol: Extracted total RNA was labeled with Cy5 using the 3D-Gene miRNA labeling kit (Toray, Kamakura, Japan)

growth_protocol: Serum was immediately stored at -80℃ after collected.ど

extract_protocol: Total RNA was extracted from the serum-EVs, cell-EVs using 3D-Gene RNA extraction reagent (Toray, Kamakura, Japan) according to the manufacturer’s instructions.

treatment_protocol: Serum-EVs were extracted using exoRNeasy Serum / Plasma kit (Qiagen), and cell-EVs were extracted using size-exclusion chromatography (SEC) column. Collected EVs were stored at -80℃.

value: cell EVs

value: oral squamous cell carcinoma

tag: phenotype

value: radioresistant (cell culture)


title: IR-SAS-R EVs

description: exo-SAS-R

tag: tissue


title: HUDEP1_L_B

value: Umbilical cord blood

value: Late (_L)

value: Cell line HUDEP-1


title: HUDEP1_L_A