GSM1414509: HeLaS3 emx1h ChIP-seq; Homo sapiens; ChIP-Seq
Sample information curated by ChIP-Atlas
Antigen
Antigen Class
TFs and others
Antigen
GFP
Cell type
Cell type Class
Uterus
Cell type
HeLa
Primary Tissue
Cervix
Tissue Diagnosis
Adenocarcinoma
Attributes by original data submitter
Sample
source_name
HeLaS3_emx1 sgRNA
cell line
HeLaS3
transfected with
emx1 sgRNA
chip antibody
GFP antibody
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Cells were cross-linked with 1% formaldehyde for 5 min at room temperature and formaldehyde was then inactivated by the addition of 125 mM glycine for 5 min at room temperature. Cells were rinsed twice with cold PBS, scraped, and collected in cold PBS, and centrifuged at 550gfor 5 min at 4 °C. The cell pellet (10^6 cells) was resuspended in 1ml ChIP cell lysis buffer (20 mMTrisHCl pH 8.0, 85 mMKCl, 0.5% NP40, 1 mM PMSF), incubated on ice for 20 minutes and then centrifuged at 550 g for 5 min at 4 °C. The pellet was resuspended in 0.2ml nuclei lysis buffer (50 mMTrisHCl pH 8.0, 10 mM EDTA, 0.1% SDS, 1 mM PMSF) and incubated on ice for 15 minutes. The lysate was sonicated (30 min total time, 30 s on, 30 s off, in 6 rounds of 5 min) in a Bioruptor (Diagenode). The lysate was centrifuged in a microfuge at 4 °C at 18,000 g for 30 min. Supernatant was collected, and 10 µl was saved as input. A single-chain anti-GFP nanobody was prepared as described before [Zhou ZX, Zhang MJ, Peng X, et al.Genome research 2013; 23: 705-715], and cross-linked to NHS-activated Sepharose beads(NERCB). 30 µl GBP beads were blocked with 500 µl 2XChIP dilution buffer (20 mMTrisHCl pH 8.0,200 mMNaCl,0.133% SDS, 1.33% Trition X-100), 450 µl H2O and 50 µl 10% BSA with rotation at 4 °C for 1 hour. 200 µl sonicated chromatin was pre-cleaned with 30 µl Sepharose beads (NERCB) with rotation at 4 °C for 1 hour in 600 µl total volume (200 µl sonicated chromatin, 300 µl 2×ChIP dilution buffer, 12 µl 100 mM PMSF and 88 µl H2O). GBP beads were resuspended in 600 µl pre-cleaned chromatin lysate to immunoprecipitate overnight at 4 °C. The next day, GBP beads were washed with 1 ml of low salt washing buffer(20 mMTrisHCl pH 8.0,150 mMNaCl,0.1% SDS, 1% Trition X-100, 2 mM EDTA), high salt washing buffer(20 mMTrisHCl pH 8.0, 500 mMNaCl, 0.1% SDS, 1% Trition X-100, 2 mM EDTA), LiCl wash buffer (20 mMTrisHCl pH 8.0, 0.25 mMLiCl, 1% NP40, 1% deoxycholate, 1mM EDTA), and TE+0.2% Trition X-100, respectively. Each wash was accomplished with rotation at 4 °C for 15 minutes. Chromatin was eluted at 65 °C for 2 hours in 200 µl ChIP elution buffer (10 mMTrisHCl pH 8.0,1 mMEDTA,0.2 mMNaCl, 0.4% SDS). Both input and immunoprecipitation samples were reversed cross-links at 65 °C overnight in 400 µl total volume with 300 mMNaCl. DNA was extracted with phenol/chloroform and precipitated with ethanol. Finally, the DNA was dissolved in 100 µl H2O. Two biological repeat libraries for each -sgRNA control, sgRNA-emx1, and sgRNA-egfa-t1samples, were prepared and sequenced. 30-40 ng of immunoprecipitated DNA were used to prepare sequencing libraries for Illumina via NEBNext® DNA Library Prep Master Mix Set (NEB #E6040L) according to manufacturer’s protocol. DNA fragments around 200 bp were isolated by Agencourt AMPure XP(Beckman coulter) and PCR amplified with NEBNextmultiplexoligos for Illumina(#E7335,#E7500). The libraries were sequenced by Illumina Hi-seq 2000 at the Shenzhen Huada Genomics Institute (China) for single-end 50bp reads.