GSM5570287: Hap1 WT H3K36me3 ChIPseq; Homo sapiens; ChIP-Seq
Cells were fixed in 1% formaldehyde; 50mM Hepes-KOH; 100mM NaCl; 1 mM EDTA; 0,5 mM EGTA. Cell lysis was performed in LB1 buffer with final pH 7,5 (50 mM Hepes-KOH; 140mM NaCl; 1mM EDTA, 10% Glycerol, 0.5% NP-40; 0,25% Triton X-100; Proteinase inhibitor) for 20 minutes at 4°C. Subsequently, nuclei were lysed using LB2, pH 8,0 (10mM Tris-HCl; 200 mM NaCl; 1mM EDTA; 0,5mM EGTA; Proteinase inhibitor) for 10 minutes at 4°C. Pellets were resuspended in LB3 pH8,0 (10mM Tris-HCl; 100mM NaCl; 1 mM EDTA; 0,5 mM EGTA; 0.1% Na-Deoxycholate; 0.5% N-lauroylsarcosine; Proteinase inhibitor). Cross-linked chromatin was sheared (400-800 bp) using a Covaris S2 with Tube and Caps (Covaris, 520048), using the following settings: duty cycle: 10%, intensity: 4, cycles per burst: 200 time 40 seconds with 20 cycles. Chromatin precipitation was performed overnight using antibody-bound (CTCF 5 μl, SMC1 10 μl, Igg 10 μl per IP) proteinA coupled DynaBeads (Invitrogen). Elution and decrosslinking was performed overnight at 65°C in EB pH 8,0 (50mM Tris-HCl; 10mM EDTA; 1% SDS). Samples were treated with Proteinase K and RNAseA for 2 hours. DNA was isolated using phenol extraction and ethanol precipitation. Library preparation was done using a KAPA Library preparation kit using the manufacturer's protocol.