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Sample GSM4547521

Query DataSets for GSM4547521

Status

Public on Aug 13, 2020

Title

H3K27me3_ChIP-seq_E35A-H2B_1

Sample type

SRA

Source name

3T3-L1

Organism

Mus musculus

Characteristics

cell type: mouse fibroblasts
sample type: H3K27me3_E35A-H2B_1

Treatment protocol

For H2B-Ser36p ChIP-seq, 3T3-L1 cells were cultured in 15 cm dishes, and treated with or without 10 uM of BYK204165 for 2 hours before collection. For H3K27me3 ChIP-seq, 3T3-L1 cells expressing FLAG-tagged wild-type or mutant (E35A) histone H2B were seeded in 15 cm dishes.

Growth protocol

3T3-L1 cells were obtained from the American Type Cell Culture (ATCC, CL-173) and were regularly verified as mycoplasma-free. They were cultured in DMEM (Cellgro, 10-017-CM) supplemented with 10% fetal bovine serum (Sigma, F8067) and 1% penicillin/streptomycin.

Extracted molecule

genomic DNA

Extraction protocol

3T3-L1 cells were seeded at ~3 x 106 cells per 15 cm diameter plate and treated as described above. The cells were cross-linked with 1% paraformaldehyde in PBS for 10 minutes at 37°C and quenched in 125 mM glycine in PBS for 5 minutes at 4°C. The cells were then collected and lysed in Farnham lysis buffer (5 mM PIPES pH 8.0, 85 mM KCl, 0.5% NP-40, 1 mM DTT, 1x phosphatase inhibitor cocktail, and 1x complete protease inhibitor cocktail). A crude nuclear pellet was collected by centrifugation, resuspended in lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl, pH 7.9, 1 mM DTT, 1x phosphatase inhibitor cocktail, and 1x complete protease inhibitor cocktail), and incubated on ice for 10 minutes. The chromatin was sheared at 4°C by sonication using a Bioruptor UC200 at the high setting for thirteen cycles of 30 seconds on and 30 seconds off to generate chromatin fragments of ~300 bp in length. The soluble chromatin was diluted 1:10 with dilution buffer (20 mM Tris-HCl, pH 7.9, 0.5% Triton X-100, 2 mM EDTA, 150 mM NaCl, 1 mM DTT, 1x phosphatase inhibitor cocktail, and 1x complete protease inhibitor cocktail) and pre-cleared with protein A Dynabeads. The pre-cleared supernatant was used in immunoprecipitation reactions with antibodies against the factor of interest or with rabbit IgG as a control, and incubated overnignt at 4°C. The protein A Dynabeads was then added and incubated at 4°C for 2 hours. The immunoprecipitated material was washed once with low salt wash buffer (20 mM Tris-HCl, pH 7.9, 2 mM EDTA, 125 mM NaCl, 0.05% SDS, 1% Triton X-100, 1x phosphatase inhibitor cocktail, and 1x complete protease inhibitor cocktail), once with high-salt wash buffer (20 mM Tris-HCl, pH 7.9, 2 mM EDTA, 500 mM NaCl, 0.05% SDS, 1% Triton X-100, 1x phosphatase inhibitor cocktail, and 1x complete protease inhibitor cocktail), once with LiCl wash buffer (10 mM Tris-HCl, pH 7.9, 1 mM EDTA, 250 mM LiCl, 1% NP-40, 1% sodium deoxycholate, 1x phosphatase inhibitor cocktail, and 1x complete protease inhibitor cocktail), and once with 1x Tris-EDTA (TE). The immunoprecipitated material was eluted in elution buffer (100 mM NaHCO3, 1% SDS) and was then digested with proteinase K and RNase H to remove protein and RNA, respectively. The immunoprecipitated genomic DNA was then extracted with phenol:chloroform:isoamyl alcohol and precipitated with ethanol. .
After purification, 10 ng of ChIP’d DNA for each condition was used to generate libraries for sequencing. Briefly, the DNA was end-repaired and a single “A”-base overhang was added using the Klenow fragment of E. coli DNA polymerase. The A-modified DNA was ligated with Ilumina sequencing adaptors using the Illumina TruSeq DNA Sample Prep Kit. The ligated DNA (250 ± 25 bp) was size-selected by agarose gel electrophoresis and extraction, amplified by PCR, and purified using AmPure beads (Beckman Coulter). The final libraries were subjected to QC (size, purity, adapter contamination) and sequenced using an Illumina HiSeq 2000 per the manufacturer’s instructions.

Library strategy

ChIP-Seq

Library source

genomic

Library selection

ChIP

Instrument model

Illumina HiSeq 2000

Description

ChIP-enriched DNA from 3T3-L1 cells ectopically expressing E35A mutant histone H2B

Data processing

The ChIP-seq reads were aligned to the mm10 mouse reference genome using the Bowtie software package (Langmead et al, 2009) .
Mapped reads were further converted to (1) “bed” files for later Metagene and read-density analyses and (2) “wiggle” files counting reads in non-overlapping 200-bp windows across the genome for presentation as genome browser tracks by using the BEDTools software package (Quinlan et al, 2010).
Genome_build: mm10
Supplementary_files_format_and_content: bigwig

Submission date

May 12, 2020

Last update date

Aug 13, 2020

Contact name

W. Lee Kraus

E-mail(s)

lee.kraus@utsouthwestern.edu

Organization name

UT Southwestern Medical Center

Street address

5323 Harry Hines Blvd.