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| Status |
Public on May 02, 2019 |
| Title |
CD8-Human-Channel2 |
| Sample type |
SRA |
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| Source name |
CD8+ T Cells
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| Organism |
Homo sapiens |
| Characteristics |
cell type: CD8+ T Cells
bead concentration: 1600
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| Growth protocol |
Cryopreserved human isolated bone marrow CD34+ stem/progenitor cells, and isolated peripheral blood CD4+ T cells, CD8+ T cells, CD14+ (Monocytes), CD19+ (B cells) and CD56+ (NK) cells were purchased from Allcells. Cells were quickly thawed in a 37°C water bath, rinsed with culture medium (IMDM medium supplemented with 10% FBS and 1% Pen/Strep) and then treated with 0.2 U/μL DNase I (Thermo Fisher Scientific) in 10 mL of culture medium at 37°C for 30 min. After DNase I treatment, cells were washed with medium once and then twice with ice cold 1x PBS + 0.1% BSA. Cells were then filtered with a 35 μm cell strainer (Corning) and cell viability and concentration were measured with trypan blue on the TC20 Automated Cell Counter (Bio-Rad). Cell viability was greater than 80% for all samples.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Lysis was performed simultaneously with tagmentation. Washed and pelleted cells were resuspended in Whole Cell Tagmentation Mix containing 0.1% Tween-20, 0.01% Digitonin, 1X PBS supplemented with 0.1% BSA, ATAC Tagmentation Buffer and ATAC Tagmentation Enzyme (parts of the SureCell ATAC-Seq Library Prep Kit - 17004620, Bio-Rad). Cells were mixed and agitated on a ThermoMixer for 30 min at 37°C. Tagmented cells were kept on ice prior to encapsulation.
Tagmented cells were loaded onto a ddSEQ Single-Cell Isolator (12004336, Bio-Rad). Single-cell ATAC-Seq libraries were prepared using the SureCell ATAC-Seq Library Prep Kit (17004620, Bio-Rad) and SureCell ddSEQ Index Kit (12009360, Bio-Rad). Bead barcoding and sample indexing was performed in a C1000 Touch™ Thermal cycler with a 96-Deep Well Reaction Module (1851197, Bio-Rad): 37°C for 30 min, 85°C for 10 min, 72°C for 5 min, 98°C for 30 sec, 8 cycles of 98°C for 10 sec, 55°C for 30 sec, and 72°C for 60 sec, and a single 72°C extension for 5 min to finish. Emulsions were broken and products cleaned up using Ampure XP beads (A63880, Beckman Coulter). Barcoded ATAC amplicons were further amplified using a C1000 Touch™ Thermal cycler with a 96-Deep Well Reaction Module: 98°C for 30 sec, 6-9 cycles (cycle number depending on the cell input, Section 4 Table 3 of the User Guide) of 98°C for 10 sec, 55°C for 30 sec, and 72°C for 60 sec, and a single 72°C extension for 5 min to finish. PCR products were purified using Ampure XP beads and quantified on an Agilent Bioanalyzer (G2939BA, Agilent) using the High-Sensitivity DNA kit (5067-4626, Agilent). Libraries were loaded at 1.5 pM on a NextSeq 550 (SY-415-1002, Illumina) using the NextSeq High Output Kit (150 cycles; 20024907, Illumina) and sequencing was performed using the following read protocol: Read 1 118 cycles, i7 index read 8 cycles, and Read 2 40 cycles. A custom sequencing primer is required for Read 1 (16005986, Bio-Rad; included in the kit).
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| Library strategy |
ATAC-seq |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
NextSeq 550 |
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| Data processing |
Barcode sequences were parsed and trimmed from FASTQs using custom python scripts implemented in the bap-barcode module.
Paired-end reads were aligned to hg19 using bwa.
Duplicate reads and all downstream processing was performed using the bead-based scATAC-seq processing (bap) tool.
Samples of common biological origin were pooled into single ranged summarized experiment obejcts using bap and shown below.
Genome_build: hg19
Supplementary_files_format_and_content: Decomposed counts matrix. CountsData is indexed by peak (row), cell (column), and count where the Bed file and CellData provide annotations for rows and columns. Barcode translate file maps raw sequence barcode (many) to cell barcode (one) from the bap output. The BarcodeLogic Excel file contains the information needed to parse the cell barcode from the sequencing data.
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| Submission date |
Dec 10, 2018 |
| Last update date |
May 02, 2019 |
| Contact name |
Caleb Lareau |
| E-mail(s) |
[email protected]
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| Organization name |
Memorial Sloan Kettering
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| Street address |
417 E 68th St, Zuckerman - ZRC 1132
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| City |
New York |
| State/province |
New York |
| ZIP/Postal code |
10065 |
| Country |
USA |
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| Platform ID |
GPL21697 |
| Series (2) |
| GSE123578 |
High-throughput combinatorial indexing enables scalable single-cell chromatin accessibility profiling [Blood] |
| GSE123581 |
Droplet-based combinatorial indexing for massive-scale single-cell chromatin accessibility |
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| Relations |
| BioSample |
SAMN10574876 |
| SRA |
SRX5124199 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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