GSM1856048: Input PHBsiRNA ChIP-Seq; Homo sapiens; ChIP-Seq
Sample information curated by ChIP-Atlas
Antigen
Antigen Class
Input control
Antigen
Input control
Cell type
Cell type Class
Pluripotent stem cell
Cell type
ES cells
NA
NA
Attributes by original data submitter
Sample
source_name
H3.3B-HA tagged human embryonic stem cells
cell type
human embryonic stem cells
passages
42-46
chip antibody
none
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Cells were cross-linked with 1% formaldehyde (Sigma) for 15 minutes at 37oC and then quenched with 0.125 M glycine (Sigma) for 5 minutes at room temperature. Cells were scraped and transferred into Eppendorf tubes with cold PBS supplemented with 1×PIC, and then centrifuged to remove supernatant. Cell pellet was suspended in 1% SDS FA cell lysis buffer comprised of 50 mM HEPES- KOH (Sigma), pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, and 1% SDS supplemented with 1×PIC. After rotating for 15 minutes at 4oC, the suspension mixture was centrifuged at 15,000×g for 45 minutes and supernatant was discarded. The pellet was suspended in 1 ml of 0.1% SDS FA cell lysis buffer supplemented with 1×PIC and 1× PMSF, and then sonicated at 50% amplitude (30S ON, 30S OFF) for 40 cycles using Sonics (UibraCELL) at 4oC, followed by centrifugation at the maximum speed for 12 minutes in a cold room. One percent of the supernatant was used as the input. The remaining supernatant was precleaned and incubated with specific antibody overnight at 4oC. On the next day, 100 μl of the Protein A and Protein G Dynabead mixture were employed to pull down antibody-precipitated protein-DNA complexes for 15 minutes at room temperature, followed by gentle wash with a low salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 150 mM NaCl, 20 mM Tris-HCl; pH8.1), a high salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 500 mM NaCl, 20 mM Tris-HCl; pH8.1), a LiCl wash buffer (0.25M LiCl, 1% NP40, 1% deoxycholate, 1 mM EDTA, 10 mM Tris-HCl; pH8.1), and a TE buffer (10 mM Tris-HCl, 1 mM EDTA; pH8.1). Chromatin was dissociated from proteins by 200 μl freshly prepared elution buffer (1% SDS, 0.1 M NaHCO3) and decross-linked by adding 8 μl NaCl and 1 μl of 10 μg/μl RNase A (Roche) at 65oC for no longer than 16 hours, followed by the treatment with 4 μl of 0.5 M EDTA, 8 μl of 1 M Tris-HCl (pH6.5) and 1 μl of 20 mg/ml protease K (Sigma). DNA was purified by a QIAquick PCR Purification Kit as instructed. Thirty ng of DNA was used for CHIP-Seq (Shanghai Biotechnology Corporation). The ChIP-enriched DNA samples were treated with end-repair of the DNA, adding ‘‘A” bases to the DNA, ligating sequencing adapters to DNA fragments, amplifying adapter-modified DNA by PCR and gel purification for ChIPseq using NEBNext Ultra DNA Library Prep Kit for Illumina. Purified libraries were sequenced on Illumina HiSeq2500 platforms.