title: GSM8601935: e2_dmso_24hr_chip_aHA-CST_rep4_20; Mus musculus; ChIP-Seq
library_construction_protocol: For HA CHIP 30M treated cells were first fixed with DSG for 50 followed by  1% formaldehyde fixation at room temperature (RT) for 10 minutes and quenched with 125mM glycine 5 mins at RT.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 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 NovaSeq 6000. PE x100