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| Status |
Public on Jun 20, 2019 |
| Title |
Validation_Exp1 |
| Sample type |
SRA |
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| Source name |
peripheral blood mononuclear cells
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| Organism |
Homo sapiens |
| Characteristics |
cell type: peripheral blood mononuclear cells
bead concentration: 200
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| Growth protocol |
Cryopreserved human peripheral blood mononuclear 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). A library of high-diversity oligonucleotides were spiked into the droplet mixture before cell loading to provide an orthogonal validation for bead loading.
SureCell scATAC-seq
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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 modeule.
Paired-end reads were aligned to hg19 reference genome using BWA
A custom python script was used to extract random oligonucleotide tags from the sequencing libraries.
Duplicate reads and all downstream processing, including inference of a bead merging statistic was performed using the bead-based scATAC-seq processing (bap) tool. Two versions of the summary statistics are presented for bap 1 (v0.5.0) and bap2 (v0.6.0)
Genome_build: hg19
Supplementary_files_format_and_content: Summary statistics regarding bead merging computed by bap or bap2 and corresponding shared oligo tag abundances per barcode pair
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| Submission date |
Jun 20, 2019 |
| Last update date |
Jun 21, 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) |
| GSE123581 |
Droplet-based combinatorial indexing for massive-scale single-cell chromatin accessibility |
| GSE133071 |
High-throughput combinatorial indexing enables scalable single-cell chromatin accessibility profiling [Validation] |
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| Relations |
| BioSample |
SAMN12098777 |
| SRA |
SRX6098121 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3899387_Validation_Exp1.bap1.sum_stats.csv.gz |
2.9 Mb |
(ftp)(http) |
CSV |
| GSM3899387_Validation_Exp1.bap2.sum_stats.csv.gz |
7.7 Mb |
(ftp)(http) |
CSV |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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