title: GSM8826151: RNAPII_Dot1l_KO_rep2; Mus musculus; ChIP-Seq
library_construction_protocol: 10 million CH12F3 cells cultured in 10mL of media were cross-linked with 1% formaldehyde for 8 minutes at room temperature, washed and resuspended in RIPA buffer and sonicated with Covaris E220 for 10 min with 10% Duty Factor, 140W PIP and 200 CBP and clarified by centrifugation. S2/Kc167 chromatin was prepared by crosslinking cells in 1X PBS containing 1% formaldehyde at RT for 20 min and quenched with 125 mM glycine for 5 min, washed and resuspended in 130 μL of RIPA buffer and fragmented using Covaris E220 for 150s with 140W PIP, 20% DF and 200 CPB and clarified by centrifugation. For spike-in, 1μg of S2/Kc167 chromatin was spiked to 24μg of CH12-F3 chromatin (1:24 ratio) before IP. Samples were pre-cleared with 20μL of Dynabeads Protein G for 1h (4°C). For the IP, 30μL Dynabeads Protein G were coupled with the target Ab (amounts as per manufacturer instructions) and 2μg of a spike-in Ab against Drosophila H2Av for 30 min and blocked 1h (4°C) with BT buffer. IP was performed by overnight incubation of the pre-cleared chromatin with blocked Ab-beads complexes. Beads were then washed and immunocomplexes were eluted and incubated overnight with proteinase K at 65°C. DNA was purified using QIAquick PCR purification kit (Qiagen), and DNA resuspended in 50μl Tris-HCl (pH 8). Libraries were prepared using the KAPA HyperPrep library kit (Roche Diagnostics) as per manufacturer's instructions. Normalization of the samples quantities was done after quantification of the ligation products by qPCR and library size distribution was assessed on a 2100 bioanalyzer.