GSM1916146: ES Gm15446 ChIPseq R2; Mus musculus; ChIP-Seq
Sample information curated by ChIP-Atlas
Antigen
Antigen Class
TFs and others
Antigen
Epitope tags
Cell type
Cell type Class
Pluripotent stem cell
Cell type
ES cells
NA
NA
Attributes by original data submitter
Sample
source_name
Embryonic stem cell (ES3)
strain
C57BL/6
cell type
Embryonic stem cells (ES3)
genotype/variation
overexpressing HA-tagged Gm15446
chip antibody
HA (Covance, MMS-101P)
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Cells were harvested, washed with Episerf, fixed in 2mL per 1x107 cells (10 min in 1% formaldehyde), quenched with glycine in 10mL (at 125 mM final), washed three times with PBS, and pelleted. Each pellet containing 1x107 cells was lysed, resuspended in 1 mL of sonication buffer on ice (10 mM Tris at pH 8, 1 mM EDTA, 0.1% SDS, and protease inhibitors), transferred to TC 12x12 tubes (Covaris), and sonicated (Covaris settings: 20 min, 5% duty cycle, 140W, 200 cycles). Sonication was assessed by reverse cross-linking (65oC, RNAse A at 1µg/µL, overnight), followed by DNA extraction. Fragment size (between 200-400bp) was checked on a Bioanalyzer (Agilent 2100). Immunoprecipitations were performed with chromatin from 4x107, with Dynabeads (Protein G, ThermoFisher) in IP buffer (10 mM Tris at pH 8, 1 mM EDTA, 0.1% SDS, 150 mM NaCl, 10% Triton X-100, and protease inhibitors) overnight. Chromatin was reversed cross-linked (65oC, Proteinase K at 400ng/µL, overnight) and DNA was extracted. Single-end libraries were prepared and sequenced in 100bp reads runs according to Illumina instructions.