GSM2966438: sgPKCi_Input; Homo sapiens; MeDIP-Seq
Total genomic DNA from samples was extracted using QIAamp DNA mini kit (QIAGEN) and eluted in DDW. 20 mg of total genomic DNA was diluted in 50 ml TE and sonicated using Covaris S2 with the following parameters (Duty 5%, Cycles/burst = 200, time=60s, 4 cycles using a microTUBE AFA Fiber Snap-Cap). Sonicated DNA was cleaned with Qiagen PCR clean UP (QIAGEN) and eluted in 30 ml of EB 2 mg of sonicated genomic DNA was used to prepare libraries using NEBNext Ultra II DNA Library Prep kit (Illumina) following manufacturer's instructions. Size-selected, end-repaired and adapter-ligated fragments (libraries) were denatured 10 min at 95C and immediately transferred to ice. 500 ng of library DNA were diluted in 500 ml of ice-cold IP buffer (20mM Tris-Cl, pH 8, 2mM EDTA, 1% Triton-X, 150 mM NaCl) with the addition of 1 mg of anti-5mC antibody (33D3, Diagenode) and incubated at 4C overnight. Same amount of library was kept aside for input sequencing and incorporated later to the Elution steps
GEO Accession
GSM2966438