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Sample GSM2544236

Query DataSets for GSM2544236

Status

Public on Mar 29, 2017

Title

H3K27ac_monocyte1

Sample type

SRA

Source name

THP-1 cells, - PMA exposure, H3K27ac ChIP

Organism

Homo sapiens

Characteristics

cell line: THP-1
pma exposure: ( - PMA )
cell type: monocytes
chip antibody: anti-H3K27ac (abcam ab4729)

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 (~10 million cells per ChIP experiment) and resuspended in growth media at 1 x 10^6 cells/mL and cross-linked with rotation at room temperature in 1% formaldehyde for 10 minutes. Cross-linking was quenched with the addition of 200 mM glycine and an additional 5 minutes of rotation at room temperature. Cross-linked cells were then spun down and resuspended in 1x RIPA lysis buffer, followed by chromatin shearing via sonication (3 cycles using a Branson sonicator: 30 seconds on, 60 seconds off; 15 additional cycles on a Bioruptor sonicator: 30 seconds on, 30 seconds off). Individual ChIP experiments were performed on pre-cleared chromatin using antibody-coupled Dynabead protein G (ThermoFisher) magnetic beads. Anti-histone H3 (acetyl K27) antibody was obtained from Abcam (ab4729), and POLR3D antibody was obtained from abcam (ab86786; Lot# GR267691-1). 3-5 ug of antibody per ChIP was coupled to 18 uL beads and rotated overnight with sheared chromatin at 40 C. Beads were then washed 5x in ChIP wash buffer (Santa Cruz), 1x in TE, and chromatin eluted in TE + 1% SDS. Cross-linking was then reversed by incubation at 65C overnight, followed by digestion of RNA (30 min RNase incubation at 37C) and digestion of protein (30 min proteinase K incubation at 45C). ChIP DNA was then purified on a minElute column (Qiagen).
Libraries were prepared for sequencing using standard Illumina protocols.

Library strategy

ChIP-Seq

Library source

genomic

Library selection

ChIP

Instrument model

Illumina HiSeq 2500

Description

ChIP-seq monocyte biological replicate 1.
Processed data file: H3K27_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