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| Status |
Public on May 02, 2019 |
| Title |
Mix-ht-sciATAC-seq-8k-input-48Tn5 |
| Sample type |
SRA |
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| Source name |
Cell line mixture
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| Organisms |
Homo sapiens; Mus musculus |
| Characteristics |
celltype: K562 and 3T3 cell line mixture
bead concentration: 5000
cell input: 8000
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| Growth protocol |
K562 (ATCC) human chronic myelogenous leukemia cells were maintained in Iscove’s Modified Dulbecco’s Medium (IMDM) (ATCC) supplemented with 10% FBS and 1% Pen/Strep. NIH/3T3 (ATCC) mouse embryonic fibroblast cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) (ATCC) supplemented with 10% Calf Bovine Serum and 1% Pen/Strep. All cell lines were maintained at 37°C and 5% CO2 at recommended density and were harvested at mid-log phase for all experiments. All suspension cells were harvested using standard cell culture procedure, and adherent cells were detached using TrypLE Express Enzyme (Gibco). After harvesting, cells were washed twice with ice cold 1x PBS (Gibco) supplemented with 0.1% BSA (MilliporeSigma). 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 90% for all samples.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
A protocol based on Omni-ATAC was followed. Briefly, washed and pelleted cells were lysed with the Omni-ATAC lysis buffer containing 0.1% NP-40, 0.1% Tween-20, 0.01% Digitonin, 10 mM NaCl, 3 mM MgCl2, and 10 mM Tris-HCl pH7.4 for 3 min on ice. The lysis buffer was diluted with ATAC-Tween buffer that only contains 0.1% Tween-20 as a detergent. Cells were collected and resuspended in Omni Tagmentation Mix containing ATAC Tagmentation Buffer and ATAC Tagmentation Enzyme that are parts of the SureCell ATAC-Seq Library Prep Kit (17004620, Bio-Rad). The Omni Tagmentation Mix was buffered with 1X PBS supplemented with 0.1% BSA. 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).
dsciATAC-seq (Barcoded-transposition 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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| Description |
N711-Sample1_S4
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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 or mm10 using bwa.
Duplicate reads and all downstream processing was performed using the bead-based scATAC-seq processing (bap) tool.
NA
Genome_build: hg19 and mm10
Supplementary_files_format_and_content: Per-sample quality-control statistics used for species-mixing inference. 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 |
May 01, 2019 |
| Last update date |
May 02, 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 |
GPL24625 |
| Series (2) |
| GSE123581 |
Droplet-based combinatorial indexing for massive-scale single-cell chromatin accessibility |
| GSE130536 |
High-throughput combinatorial indexing enables scalable single-cell chromatin accessibility profiling [human K562 and muring 3T3] |
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| Relations |
| BioSample |
SAMN11547091 |
| SRA |
SRX5773022 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3741942_Exp120-Sample1.QCstats.csv.gz |
93.1 Kb |
(ftp)(http) |
CSV |
| GSM3741942_Exp120-Sample1.barcodeTranslate.tsv.gz |
92.1 Kb |
(ftp)(http) |
TSV |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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