GSM2438485: EC-EA-3702-INPUT MIT 1; 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
embryonic stem cells
strain
C57BL/6-129
age
E3.5
cell cycle
mitotic
chip antibody
none
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Asynchronous or mitotic ESCs were crosslinked in 1% formaldehyde at room temperature (RT) for 10 minutes and quenched with 125mM glycine 5 mins at RT. 25-50M ESCs were used for the TF ChIPs and 10M for the H3K27acetylation ChIP. Cell pellets were washed twice in PBS and resuspended in 400ul lysis buffer per 20 million cells (10mM Tris pH8, 1mM EDTA, 0.5% SDS). Cells were sonicated in a bioruptor device (30 cycles 30sec on/off, high setting) and spin down 10 minutes at 4 degrees at maximum speed. Supernatants were diluted 5 times with dilution buffer (0.01%SDS, 1.1% triton,1.2mM EDTA,16.7mM Tris pH8, 167mM NaCl) and incubated with the respective antibody (2-3ug/10M cells) O/N with rotation at 4oC. Next day, protein G Dynabeads (ThermoScientific) preblocked with BSA protein (100ng per 10ul Dynabeads) were added (10ul blocked Dynabeads per 10 million cells) and incubated for 2-3 hours at 4oC. Beads were immobilized on magnet and washed twice in low salt buffer (0.1% SDS,1% triton, 2mM EDTA, 150mM NaCl, 20mM Tris pH8), twice in high salt buffer (0.1% SDS,1% triton, 2mM EDTA, 500mM NaCl, 20mM Tris pH8), twice in LiCl buffer (0.25M LiCl, 1% NP40, 1% deoxycholic acid (sodium salt), 1mM EDTA, 10mM Tris pH8) and once in TE. DNA was then eluted from the beads by incubating with 150ul elution buffer (1% SDS, 100mM NaHCO3) for 20 minutes at 65 degrees (vortex every 10min). Supernatants were collected and reverse-crosslinked by incubation at 65 degrees O/N in presence of proteinase K. After Rnase A treatment for 1hr at 37oC, DNA was purified using the Qiagen minElute kit. 6-10ng of immunoprecipitated material was used for ChIP-seq. ChIP-seq library preparation using the KAPA Hyper prep kit from KAPA Biosystems. Libraries were sequenced on an Illumina HiSeq 5000 platform on the SE50 mode.