GSM3016993: ES PCGF5 input; Mus musculus; 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
46C mES
cell type
embryonic stem cells (ES)
chip/antibody
none
genotype/variation
Wild type
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
cells were crosslinked with 2mM EGS [ethylene glycol bis(succinimidylsuccinate)] and crosslink it again with 1%formaldehyde. The cells were sonicated to achieve a chromatin sized of 200-500 bp in SDS lysis buffer containing 1 × protease inhibitor cocktails and 1 mM PMSF. Then the sonicated chromatin was dialyzed with TE buffer containing 150 mM Nacl and incubated with M2-Magnetic beads overnight at 4°C with rotation. Wash twice with TBS-T (Tween-20 at 0.05%), and wash four times with TBS at 4°C. For H2AK119ub1 and H3K27me3, chromatin was extracted in buffer A (10mM Tris-HCL PH=7.9, 1.5mM Mgcl2, 10mM KCL, 0.1% TritonX-100, protease inhibitors). The resultant chromatin was re-suspended in 200ul Buffer B (50 mM Tris–HCl, pH 7.9, 3.5mM MgCl2, 2 mM CaCl2, protease inhibitors) and digested into nucleosomes fragments at 37℃ by adding 5ul 200U/ml MNase. After that, nucleosomes fragments were crosslinked with 0.5% formaldehyde immediately. Magnetic protein A/G beads were washed and added with the recommended antibody for overnight. Immune complexes were washed and Antibody-bound chromatin was reverse-crosslinked, and the ChIPed DNA samples were purified for either ChIP-Seq. The amounts of immunoprecipitated DNA were normalized to the input. About 10 ng IP DNA and input DNA measured by Qubit Fluorometer (Invitrogen) were used to construct DNA library by using ChIP-Seq Sample Prep Kit (Illumina).