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Sample GSM2804432

Query DataSets for GSM2804432

Status

Public on Sep 24, 2018

Title

H3K27ac-Control_dup2

Sample type

SRA

Source name

pharyngeal epithelial cells

Organism

Homo sapiens

Characteristics

cell line: Detroit 562 cells
treatment: None
duplicates: replicate 2
antibody: anti-H3K27ac antibody (Active motif, reference 39138)

Treatment protocol

Detroit 562 cells were treated with LPS at 1μg/mL for 80 minutes or with fresh medium (no treatment control).

Growth protocol

Detroit 562 cells were purchased from ATCC and cultured in RPMI medium (Gibco) supplemented with 10% fetal bovine serum performance (Gibco), 100U/ml penicillin, 100ug/ml streptomycin (Gibco) and 1mM sodium pyruvate (Gibco).

Extracted molecule

genomic DNA

Extraction protocol

After treatment, cells were crosslinked with 0.75% formaldehyde for 8 minutes and cross-linking was quenched with 180mM Glycine for 10 minutes at room temperature. Cells were then lysed with cell lysis buffer (0.25% Triton X-100, 10mM EDTA, 0.1M NaCl in 10mM Tris-HCl pH8) for 20 minutes at 4°C. Nuclei were collected by centrifugation at 4,000rpm for 10 minutes at 4°C and lysed in nuclei lysis buffer (1% NP-40, 0.5% NaDOC, 0.1% SDS in PBS) for 20 minutes at 4°C. Chromatin shearing was performed using a sonicator (Bioruptor, Diagenode) on high setting for 45 cycles of 30 seconds ON/45 seconds OFF, leading to a fragment range of 100-250bp.
ChIP-seq libraries were built following the library-on-beads protocol from Wallerman et al. (Wallerman et al. 2015). Chromatin was incubated with anti-H3K27ac antibody (Active motif, reference 39138) bound to protein G magnetic beads (Dyanabeads protein G, Thermo Fisher scientific) prepared by mixing 5ug of antibody with 50uL of beads in PBS+0.5%BSA for 4 to 6 hours at room temperature. Immunoprecipitation was performed by rotation at 4°C overnight. After immunoprecipitation, samples were washed twice with nuclei lysis buffer, twice with nuclei lysis buffer complemented with 350mM NaCl, twice with LiCl/NP-40 buffer (0.25M LiCl, 1mM EDTA, 0.5% NP-40, 0.5% NaDOC in 10mM Tris-HCl pH8) and once with PBS. Libraries on beads were then built using NEBnext DNA library prep kit (New England Biolabs) according to the manufacturer’s instructions. Due to the nature of the samples, minor adjustments were done: two washes with PBS were performed in between each step of the preparation and elution of the DNA from the beads was done after adaptor ligation. ChIP samples were eluted in Elution buffer (10mM EDTA, 1% SDS in 50mM Tris-HCl pH7.5) for 4 hours at 65°C and purified with Agencourt AMPure XP beads (Beckman Coulter). PCR reaction was performed using Phusion High-Fidelity PCR Master Mix (Thermo scientific) and custom primers for multiplexing. The following program was applied: 3 minutes at 98°C; 17x(30 seconds at 98°C, 30 seconds at 65°C, 30 seconds at 72°C); 5 minutes at 72°C. Samples were loaded on a DNA1000 bioanalyzer chip (Agilent) to check the size of the libraries as well as the concentration. As a control, 2-5% of input DNA was reverse cross-linked at 65°C for minimum 6 hours and libraries were prepared using the same library preparation kit. Samples were sequenced on an Illumina NextSeq 1x76bp.

Library strategy

ChIP-Seq

Library source

genomic

Library selection

ChIP

Instrument model

Illumina NextSeq 500

Data processing

mapping of the reads to hg19 with BWA
Filtering with samtools 1.2 (samtools view -bh -F 1804 -q 20)
Peak calling with Dfilter 1.5 (run_dfilter15.sh -d=ChIP.bam -c=Input.bam -o=H3K27ac-peaks -f=bam -ks=60 -bs=100 -lpval=1) and Irreproducibility Discovery Rate (IDR) method. IDR analysis was performed between the calls from each duplicates (in Control and LPS) and peaks with an IDR<0.05 were selected, coordinates of the final peaks were extracted from the IDR analysis output by taking the overlap of the coordinates from the 2 duplicates with Bedops –intersect command.
Black listed regions were removed found in wgEncodeDacMapabilityConsensusExcludable.bed (http://hgdownload.cse.ucsc.edu/goldenpath/hg19/encodeDCC/wgEncodeMapability/) were removed from the final list of peaks using bedtools intersect command. Any peaks falling in chromosome Y was also filtered out as the cell line studied is derived from a female.
Genome_build: hg19
Supplementary_files_format_and_content: For each condition, the final reproducible peaks are given in a bed file.

Submission date

Oct 05, 2017

Last update date

May 15, 2019

Contact name

Lisa Borghini

E-mail(s)

borghinil99@gis.a-star.edu.sg

Organization name

GIS

Department

Infectious Diseases