GEO Accession viewer

NCBI > GEO > Accession Display

Not logged in | Login

GEO help: Mouse over screen elements for information.

Sample GSM2256181

Query DataSets for GSM2256181

Status

Public on Dec 01, 2016

Title

input_hgal_1

Sample type

SRA

Source name

input_hgal

Organism

Homo sapiens

Characteristics

cell type: HeLa
sgrna infected: Gal4

Growth protocol

Cells were infected with sgRNA lentiviruses for 48 hr, followed by 4 d puromycin treatment (1 μg/mL) and 1 d recovery.

Extracted molecule

genomic DNA

Extraction protocol

Genome-wide histone modifications were determined by ChIP against H3K9me3 (Abcam ab8898) on 5 million cells as described in (14). Cells were cross-linked by adding 37% formaldehyde to a final concentration of 1% into culture medium and gently shaking for 10 min at room temperature. Reaction was quenched with glycine, and cells were then washed twice with ice-cold PBS containing protease inhibitors (1mM PMSF, 1X Roche cOmplete EDTA-free cocktail). Cells were scraped off of the plate using a cell lifter and pelleted for 5 min at 2,000 rpm at 4°C. Pellet was snap-frozen in liquid nitrogen and stored at −80°C. Pellet was then thawed and resuspended in Cell Lysis Buffer (5 mM PIPES pH 8, 85 mM KCl, freshly added 1% IGEPAL) with protease inhibitors (Pierce Halt Protease Inhibitor Cocktail). Cells were then homogenized using a type B glass dounce homogenizer, pelleted, and resuspended in Nuclei Lysis Buffer (50 mM Tris-HCl pH 8, 10 mM EDTA, 1% SDS). Chromatin was sonicated in Diagenode TPX tubes using the Diagenode Bioruptor for 20 cycles and DNA was ranged from 150–700 bps as determined by gel electrophoresis. Debris was pelleted and discarded, and an aliquot was removed for Input DNA sequencing from the sonicated chromatin within the supernatant. Sonicated chromatin was then diluted 5-fold in IP Dilution Buffer (50 mM Tris–HCl pH 7.4, 150 mM NaCl, 1% IGEPAL, 0.25% deoxycholic acid, 1 mM EDTA pH 8) with protease inhibitors and pre-cleared with Life Technologies Protein G Dynabeads for 2 hr at 4°C. 5 μg of antibody was added per million cells, and samples were incubated overnight at 4°C. Antibody-bound chromatin was then collected using Life Technologies Protein G Dynabeads and washed twice with IP Dilution Buffer, twice with IP Wash Buffer 2 (100 mM Tris–HCl pH 9, 500 mM LiCl, 1% IGEPAL, 1% deoxycholic acid), and once with IP Wash Buffer 3 (100 mM Tris–HCl pH 9, 500 mM LiCl, 150 mM NaCl, 1% IGEPAL, 1% deoxycholic acid). Precipitated chromatin was then eluted for 30 min at 65°C with Elution Buffer (1% SDS, 50 mM NaHCO3). ChIP and Input DNA crosslinks were reversed by adding 5 M NaCl and heating at 65°C overnight. The following day, 10 mg/ml RNase A was added to precipitated chromatin, and chromatin was incubated for 30 min at 37°C. DNA was then recovered using Agencourt AMPure XP Beads and quantified using the Life Technologies Qubit Fluorometer.
Kapa HyperPlus

Library strategy

ChIP-Seq

Library source

genomic

Library selection

ChIP

Instrument model

Illumina HiSeq 4000

Description

processed data file: chip_counts.txt

Data processing

RNA-seq: Reads were aligned to the human genome (GRCh37) using the spliced read aligner HISAT2 v2.0.3 against an index containing SNP and transcript information (genome_snp_tran). Quantification of Ensembl build 75 genes was carried out with featureCounts using only uniquely mapped reads.
ChIP-seq: Reads were aligned to the human genome (GRCh37) using bowtie v2.2.8. Enrichment at promoter regions, which were defined as +/- 1kb of each TSS and generated from Ensembl GRCh37 build 75, were quantified using featureCounts v1.5.0-p2.
Genome_build: GRCh37

Submission date

Jul 29, 2016

Last update date

May 15, 2019

Contact name

John Liu

E-mail(s)

john.liu@ucsf.edu

Organization name

UCSF

Street address

35 Medical Center Way