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Sample GSM5526775

Query DataSets for GSM5526775

Status

Public on Sep 23, 2021

Title

U2OS_GFP(HNF4A8_control)_BMAL1_ChIPseq_2

Sample type

SRA

Source name

U2OS_GFP(HNF4A8_control)_BMAL1_ChIPseq

Organism

Homo sapiens

Characteristics

cell line: U2OS
genotype: GFP overexpressed
chip antibody: BMAL1_ChIP-seq

Extracted molecule

genomic DNA

Extraction protocol

~ 50 mg of blood perfused liver tissue were dissected and snap frozen in liquid nitrogen. Frozen liver tissues were minced with razor blades and homogenized by pushing through 18G needle followed by 21G needle to release nuclei. Nuclei were immediately crosslinked with 1% formaldehyde for 10 min at room temperature and then quenched with glycine. After two washes, the nuclei were lysed in nuclear lysis buffer (50 mM Tris-HCl pH 8.0, 1% SDS, 10 mM EDTA) containing 1x EDTA-free protease inhibitor cocktail (Roche) and kept on ice for 5 min. Immediately, the lysates were sonicated 17 times for 30 sec by using Bioruptor (Diagenode). After centrifugation at 17,000 rpm for 15 minutes at 4°C, fragmented chromatin was diluted 10-fold with IP dilution buffer (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA) supplemented with 1x EDTA-free protease inhibitor cocktail, and incubated with antibody overnight at 4°C. Dynabeads Protein G (Invitrogen cat. #10004D) was added to the chromatin and incubated for another 3 h. Afterwards, the beads were washed once with low salt wash buffer (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 2 mM EDTA) and once with high salt wash buffer (20 mM Tris-HCl pH 8.0, 500 mM NaCl, 1% Triton X-100, 0.1% SDS, 2 mM EDTA). IP elution buffer (1% SDS, 100 mM NaHCO3) was applied to the bead pellet and incubated at 30°C for 15 min. The eluate was added with NaCl to final conc. of 200 mM and reverse-crosslinked at 65°C overnight. To remove RNA and protein, RNase A and proteinase K were subsequently applied by incubating at 45°C for 1 h. Finally, DNA was purified with QIAquick PCR Purification columns. ChIP-seq experiments with human cell lines were similar. The differences are the cells were crosslinked in culture dishes with 1% formaldehyde, and then scraped off for cell lysis and chromatin fragmentation.
2 ng of each sample was prepared using the NEB Ultra DNA Library Prep Kit for Illumina following manufacturer’s instructions. Each library was dual size selected with 0.55X and 0.14X Ampure beads followed by PCR amplification with Kappa HiFi 2x PCR mix (15 cycles). PCR product was then quantitated using the Qubit (Thermo Fisher) and BioAnalyzer (Agilent BioAnalyzer 2100) to determine library quantity. Libraries were then gel purified on a 2% agarose gel to remove secondary PCR amplification artifacts, quantitated on the Qubit and loaded onto HiSeq to generate more than 20M reads per sample.

Library strategy

ChIP-Seq

Library source

genomic

Library selection

ChIP

Instrument model

Illumina HiSeq 4000

Data processing

Illumina HiSeq 150 bp reads produced by ChIP-Seqs were mapped to the mm10 or hg38 genome using Bowtie2.
Peak calling was performed with MACS2.
Genome_build: mm10, hg38
Supplementary_files_format_and_content: bigWig files were merged replicates and generated using DeepTools with RPGC normalization. Peak files were generated by MACS2.

Submission date

Aug 17, 2021

Last update date

Sep 23, 2021

Contact name

Meng Qu

E-mail(s)

[email protected]

Phone

626-898-2391

Organization name

University of Southern California