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Sample GSM2544229

Query DataSets for GSM2544229

Status

Public on Mar 29, 2017

Title

ATAC-seq_monocyte2d

Sample type

SRA

Source name

THP-1 cells, - PMA exposure

Organism

Homo sapiens

Characteristics

cell line: THP-1
pma exposure: ( - PMA )
cell type: monocytes

Treatment protocol

For differentiation of THP-1 cells, non-adherent cells were diluted to 2 x 10^5 cells/mL and grown overnight, and a final concentration of 100 nM PMA was added the following morning. THP-1 derived macrophages were collected after 72 hr exposure to PMA by aspirating media and any non-adherent cells, and incubating adherent cells with TrypLE (ThermoFisher) for 15 minutes followed by cell wash in phosphate buffer saline (PBS) buffer.

Growth protocol

THP-1 cells were obtained from ATCC (Lot # 62454382) and grown for multiple passages in T-75 flasks between 2-8 x 10^5 cells/mL in growth medium containing RPMI-1640 (Corning), 10% fetal bovine serum, and 1% penicillin streptomycin.

Extracted molecule

genomic DNA

Extraction protocol

Equal numbers of THP-1 monocytes and THP-1 derived macrophages were collected (50,000 cells per ATAC-seq experiment) and washed with 1x ice cold PBS. Cells were pelleted via centrifugation (500 xg, 5 minutes, 40C) and resuspended in cell lysis buffer (10mM Tris-HCl, pH 7.4, 10mM NaCl, 3mM MgCl2, 0.1% IGEPAL CA-630), and immediately spun down (500 xg, 10 minutes, 40C). The supernatant was then discarded, and transposition reaction carried out for 30 minutes at 37C with Tn5 transposase in transposition buffer (Illumina, cat#FC-121-1030). DNA was immediately purified on a minElute column (Qiagen), followed by PCR amplification using the NEBNext high-fidelity master mix (NEB cat#M0541) with Nextera PCR primers and barcodes. PCR amplification was monitored as described (Buenrostro et al. 2015), and gel purified to remove contaminating primer-dimer species.
Libraries were prepared for sequencing using standard Illumina protocols.

Library strategy

ATAC-seq

Library source

genomic

Library selection

other

Instrument model

Illumina HiSeq 2500

Description

ATAC-seq monocyte biological replicate 2 sequencing replicate 4.
Processed data file: ATAC_peak_edgeR_raw.txt

Data processing

Biological replicates and experimental conditions (-PMA; +PMA) were sequenced together on an Illumina HiSeq2000 (paired-end, 100) for each individual experiment type (ATAC-seq, ChIP-seq, PRO-seq), and sequencing reads trimmed using trim galore v. 0.4.0, prior to downstream sequence alignment and analyses.
Trimmed ChIP and ATAC sequencing reads were mapped to the hg19 genome using Bowtie v 2.2.4, duplicate reads were removed by Picard tools v. 1.92 and mitochondrial reads filtered before peak-calling using MACS2 v. 2.1.0.
Alignment of sequencing reads to the current hg19 annotation of human tRNA genes (gtRNAdb; (Chan and Lowe 2009; Chan and Lowe 2016)) included the upstream and downstream 100 nt flanking genomic regions.
Genome_build: hg19 (GRCh37)
Supplementary_files_format_and_content: *.txt: tDNA count files were generated by extracting reads over tRNA genes (100nt flanking) and non-tRNA genes and normalized counts determined by DESeq.
Supplementary_files_format_and_content: *.txt: ATAC and ChIP peaks were determined using MACS2 v. 2.1.0. Changes in chromatin accessibility and histone occupancy were determined by differential read count analyses (EdgeR) over merged peak lists for each factor.

Submission date

Mar 19, 2017

Last update date

May 15, 2019

Contact name

Kevin Van Bortle

Organization name

Stanford University

Department

Genetics

Lab

Michael Snyder