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| Status |
Public on May 02, 2019 |
| Title |
HumanBoneMarrow-ht-sciATAC-seq-Channel13 |
| Sample type |
SRA |
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| Source name |
Human bone marrow mononuclear cells
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| Organism |
Homo sapiens |
| Characteristics |
cell type: Human bone marrow mononuclear cells
bead concentration: 5000
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| Treatment protocol |
Bone marrow mononuclear cells were quickly thawed in a 37°C water bath, rinsed with culture medium (RPMI 1640 medium supplemented with 15% FBS and 1% Pen/Strep) and then treated with 0.2 U/μL DNase I in 10 mL of culture medium at 37°C for 30 min. After DNase I treatment, cells were washed with medium once, filtered with a 35 μm cell strainer and cell viability and concentration were measured with trypan blue on the TC20 Automated Cell Counter. Cell viability was greater than 90% for all samples. Cells were plated at a concentration of 1 x 10^6 cell/mL, rested at 37°C and 5% CO2 for 1 h and then either incubated in serum containing media (RPMI 1640 medium supplemented with 15% FBS and 1% Pen/Strep) at 37°C and 5% CO2 for 6 h (Serum stimulation) or treated with 20 ng/mL Lipopolysaccharide (LPS) (tlrl-3pelps, Invivogen) for 6 h (LPS stimulation). After stimulation, cells were washed twice with ice cold 1x PBS + 0.1% BSA and cell viability and concentration were measured with trypan blue on the TC20 Automated Cell Counter. As a control, we processed cells immediately after counting, without any incubation.
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| Growth protocol |
Cryopreserved human bone marrow mononuclear cells were purchased from Allcells.
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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.
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. The Tn5 table provides a key for the biological condition encoded by the Tn5 barcode
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| Submission date |
Dec 10, 2018 |
| Last update date |
Jun 26, 2019 |
| Contact name |
Caleb Lareau |
| E-mail(s) |
lareauc@mskcc.org
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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) |
| GSE123580 |
High-throughput combinatorial indexing enables scalable single-cell chromatin accessibility profiling [Bone marrow] |
| GSE123581 |
Droplet-based combinatorial indexing for massive-scale single-cell chromatin accessibility |
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| Relations |
| BioSample |
SAMN10574826 |
| SRA |
SRX5124160 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3507404_HumanBoneMarrow-ht-sciATAC-seq-Channel13.fragments.tsv.gz |
288.2 Mb |
(ftp)(http) |
TSV |
| GSM3507404_HumanBoneMarrow-ht-sciATAC-seq-Channel13.fragments.tsv.gz.tbi |
991.9 Kb |
(ftp)(http) |
TBI |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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