GSM6926584: INPUT-P-KO; Homo sapiens; ChIP-Seq
Sample fixation and nuclei preparation: After 48 hrs of transfection, all cells were fixed with 37% formaldehyde for 15 min at room temperature. After on, aspirated media, washed 1x with cold PBS and collected cells in 2ml of PBS/plate . The cells were spin at 850 rpm for 10 min at 4C. The supernatant was discard and resuspend the pellet in hypotonic buffer containing fresh protease inhibitors. Cells were incubated in ice for 5 min, then added 10% NP-40 to final concentration of 0.5% , and incubated on ice for 5 min. Cells were centrifuged at 1500 rpm 10 min at 4C. Discard the supernatant and pellet was resuspended in 2mll of TE buffer containing 2x protease inhibitors.. Sonication and DNA quantitation:  The nuclei was sonicated in 15 ml conical vial on cool chamber at optimal settings (5) 10 sec pulses at 60% , which needs to be determined per cell line. For this cells was 6 rounds which was 300 seconds. After sonications, samples were split into (2) 1.7 ml eppendorf tubes and was centrifuges 14,000 rpm 15 min at 4C. Samples were collected and mix in same supernatants and DNA was measures in triplicate using nanodrop. Measurements was averaged and dived the number by 1000.Samples were aliquoted in maximal amount of DNA but less the 500 ul total volume that was normalized in TE buffer. Immunoprecipitaion: To the 500 ul of aliquots, 500 ul 2x RIPA buffer containing protease inhibitors.  40ul protein A-agarose DNA bead was added using large orifice tips and rotate 30 min at 4C. Samples were spin down at 14,000 rmp for 1 min and transferred the supernatant to a new chilled tube. 1/10th volume from each tube was removed and transfer to a new tube as INPUT, and brough the volume to 500 ul and kept at -20C until needed for next day. To the remaining volume (IP),  our protein of interest H3K27Me3 (cs # 9733) was added at 1:50 dilution and let rotate overnight at 4C. Next day, 50 ul of protein A-agarose DNA bead was added and let rotate for 2 hrs.  Samples were spin at 2500 rpm at 4C for 3 min. Supernatant was removed and pellet was washed once in order of low salt, high salt, LiCL, and twice 1X TE in 1 ml volume at RT by mixing on nutator 3-5 min and spin at 2500 rpm at RT for 3 min in between each wash. GENEWIZ DataAnalysisReport