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Sample GSM2634950

Query DataSets for GSM2634950

Status

Public on May 23, 2017

Title

singles-K562-141105-90

Sample type

SRA

Source name

K562

Organism

Homo sapiens

Characteristics

cell type: cell line, chronic myelogenous leukemia, untreated

Treatment protocol

Growth protocol

Extracted molecule

genomic DNA

Extraction protocol

Single cells were captured using the C1 Single-Cell Auto Prep IFC microfluidic chips. Cells were permeabilized and accessible fragments were captured using 20 µL of Tn5 transposition mix (1.5x TD buffer, 1.5 µL transposease (Nextera DNA Sample Prep Kit, Illumina), 1x C1 Loading Reagent with low salt (Fluidigm), and 0.15% NP40) at 30 minutes at 37°C.
In a 96-well plate, 10 µL of harvested libraries were amplified in 50 µL PCR for an additional 14 cycles (1.25 µM custom Nextera dual-index PCR primers (Supplementary Table 1) in 1x NEBnext High-Fidelity PCR Master Mix) using the following PCR conditions: 72°C for 5min; 98°C for 30 s; and thermocycling at 98°C for 10 s, 72°C for 30 s, and 72°C for 1 min. The PCR products were pooled creating a final volume of ~4.8 mL. The pooled library was purified on a single MinElute PCR purification column (Qiagen) yielding libraries at an approximate concentration of ~1 µM. Libraries were quantified using qPCR prior to sequencing.
scATAC-seq

Library strategy

ATAC-seq

Library source

genomic

Library selection

other

Instrument model

Illumina HiSeq 2000

Description

K562 (ATCC) chronic myeloid leukemia cells were maintained in Iscove’s modified Dulbecco’s medium (IMDM) containing 10% FBS (HyClone, Thermo Scientific) and 1% Penicillin Streptomycin (Pen/Strep).

Data processing

Adapter sequences were trimmed from FASTQs using custom python scripts to enable mapping fragments with sequences containing adapters. Paired-end reads were aligned to hg19 using BOWTIE2 using the parameter –X2000 allowing fragments of up to 2 kb to align. Duplicates were removed using PICARD tools. Reads were subsequently filtered for alignment quality of >Q30 and were required to be properly paired. Reads mapping to the mitochondria, unmapped contigs and chromosome Y were removed and not considered.
Genome_build: hg19
Supplementary_files_format_and_content: Peaks were called on single cell ATAC-seq data from this series as well as GSE65360 and GSE74310 using MACS2. Peak summits were called on merged bamfiles from each different celltype, fixed to 500bp around the summit, and then combined. Fragments with either end mapping inside the peak were counted for each cell for each peak.

Submission date

May 22, 2017

Last update date

May 15, 2019

Contact name

William J Greenleaf

E-mail(s)

[email protected]

Organization name

Stanford University

Department

Genetics