title: GSM3106292: EC-EA-3702-INPUT_ESC_2I; Mus musculus; ChIP-Seq
library_construction_protocol: Cells were crosslinked in 1% formaldehyde at RT for 10 minutes and quenched with 125mM glycine for 5 mins at RT. 50 million cells were used for KLF4 ChIPs and 10 million for H3K27acetylation ChIP. Cell pellets were washed twice in PBS and resuspended in 400ul lysis buffer (10mM Tris pH8, 1mM EDTA, 0.5% SDS) per 20 million cells. Cells were sonicated in a bioruptor device (30 cycles 30sec on/off, high setting) and spun down 10 minutes at 4°C 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) (KLF4 R&D #3158, H3K27ac  ab4729) O/N with rotation at 4°C. 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 4°C. Beads were immobilized on a 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°C (vortexing every 10min). Supernatants were collected and reverse-crosslinked by incubation at 65°C O/N in presence of proteinase K. After RNase A treatment for 1hr at 37°C, DNA was purified using the minElute kit (Qiagen). 6-10ng of immunoprecipitated material was used for ChIP-seq library preparation using the KAPA Hyper prep kit (KAPA Biosystems). Libraries were sequenced on an Illumina HiSeq 2500 platform on SE50 mode.
attributes.tag: GEO Accession
attributes.value: GSM3106292