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Sample GSM4784079 Query DataSets for GSM4784079
Status Public on Apr 02, 2021
Title scATAC-seq of leukapheresis-purified, 0.01% digitonin permeabilized, magnetic bead anti-CD15-depleted PBMCs
Sample type SRA
 
Source name peripheral blood mononuclear cells
Organism Homo sapiens
Characteristics donor_id: HMN85396
donor_source: BioIVT
purification_type: leukapheresis
preparation_type: permeabilized cells
preparation_conditions: 0.01% digitonin
cleanup_type: Magnetic Bead
cleanup_conditions: anti-CD15 Depletion
library_type: 10x scATAC-seq
library_type: 10x scATAC-seq
Extracted molecule genomic DNA
Extraction 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,
 
Library strategy ATAC-seq
Library source genomic
Library selection other
Instrument model Illumina NovaSeq 6000
 
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
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).
 
Submission date Sep 15, 2020
Last update date Apr 02, 2021
Contact name Allen Institute For Immunology
E-mail(s) xiaojun.li@alleninstitute.org
Phone 2065487135
Organization name Allen Institute
Street address 615 Westlake Ave N, Seattle, WA
City Seattle
State/province Washington
ZIP/Postal code 98109
Country USA
 
Platform ID GPL24676
Series (1)
GSE158013 Simultaneous trimodal single cell measurement of transcripts, epitopes, and chromatin accessibility using TEA-seq
Relations
BioSample SAMN16170818

Supplementary file Size Download File type/resource
GSM4784079_X032-AP0C1W4_leukopak_perm-cells_neudep-bead_ds125M_20kb-window-matrix.h5 58.6 Mb (ftp)(http) H5
GSM4784079_X032-AP0C1W4_leukopak_perm-cells_neudep-bead_ds125M_filtered-fragments.tsv.gz 304.9 Mb (ftp)(http) TSV
GSM4784079_X032-AP0C1W4_leukopak_perm-cells_neudep-bead_ds125M_filtered-metadata.csv.gz 1.1 Mb (ftp)(http) CSV
Raw data not provided for this record
Processed data provided as supplementary file

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