ChIP-Seq input control of rat, cell line CVCL_0351, replicate 3
10 mioINS1E cells are used per IP. Cells were crosslinked with 1% formaldehyde for 10 min at room temperature, crosslinking was stopped by addition of 2M Glycine (0.125M final concentration) at room temperature for 5 min. Cells are washed twice in cold 1x PBS. Cells are lysed into ice-cold lysis buffer (50 mM Tris-HCl, pH 8.1, 1% SDS, 10 mM EDTA, 1X protease inhibitors) for 1h in ice. Following cell lysis, the samples were sonicated (Bioruptor Plus, Diagenode) to generate fragments of average length of 300-100 base pairs. Cellular debris were removed by centrifugation at 4C for 10 min (10,000 g), 10 % of the lysate was stored as the source of Input and the remaining lysate was diluted 8 times in dilution buffer (final concentration: 16.7 mM Tris-HCl, pH 8.1, 0.01 SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM NaCl, protease inhibitor), in presence of 70 l of anti-HA Magnetic beads were then incubated at 4C with rotating for overnight.Beads were washed extensively at 4C in Low salt buffer (20mM Tris-HCl, pH 8.1, 0.1% SDS, 1%Triton X-100, 2mM EDTA, 150 mM NaCl), in High salt buffer (20mM Tris-HCl, pH 8.1, 0.1% SDS, 1%Triton X-100, 2mM EDTA, 500mM NaCl), and in LiCl buffer (10mM Tris-HCl, pH 8.1, 250mM LiCl, 1% NP-40, 1% sodium deoxycholate, 1mM EDTA), and finally in 1ml of TE buffer (10mM Tris-HCl, pH 8.0, 1mM EDTA). The bound chromatin was released from the beads by vortexing at room temperature in 200 l elution buffer (1% SDS and 100 mM NaHCO3). 1 l of 10mg/ml RnaseA and 5M NaCl (200mM final concentration) was added to the eluate and incubated O/N at 65C, and then treated with Proteinase K for 1 hr at 55C; DNA was purified using QIAGEN PCR purification kit.