title: GSM6050620: R1E_Ctr_Input; Mus musculus; ChIP-Seq
library_construction_protocol: Briefly, 40 million cells were fixed with 1% formaldehyde for 10 min at room temperature. Chromatin fraction was prepared step by step using Lysis buffer 1 (50 mM Hepes-KOH, pH 7.5, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100), Lysis buffer 2 (10 mM Tris-HCl, pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA) and Lysis buffer 3 (10 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% Na-Deoxycholdate, 0.5% N-lauroylsarcosine). All the buffers were freshly supplemented with protease inhibitor cocktails (Roche, 4693116001) and 1 mM fresh PMSF. Chromatin fraction was sonicated with a Bioruptor® Plus (Diagenode) to shear DNA to a size of 300-500 bp. 5 μg of antibody and 50 ul Protein G beads (Genscript，L00664-25) were used to immunoprecipitate DNA-protein complex. After washing 5 times with Wash buffer (50 mM Hepes-KOH, pH 7.6, 500 mM LiCl, 1 mM EDTA, 1% NP-40 and 0.7% Na-Deoxycholdate), the DNA-protein complex was eluted in TES (10 mM Tris-HCl+1% SDS). After reverse-cross linking and DNA purification, the DNA was quantified using QubitTM 3.0 (Thermo Fisher Scientific, Q33216). 30 ng ChIPed DNA was sent to Novogen for library construction and sequencing. (1) The DNA fragments are repaired, dA-tailed.(2) The DNA fragments with A tail are ligated to sequencing adaptors(3) The final DNA library is obtained by size selection and PCR amplification. After the construction of the library, the initial quantification was done with Qubit 2.0, and the library is diluted to 1ng/l. Then the insertion size of the library is detected with NGS3K. If meeting the expectation, the accurate concentration of the library is quantified by Q-PCR (library effective concentration > 2nM) to ensure the accurate molar amount that will pooled for sequencing.