GSM5808359: EV input DNA rep2; Homo sapiens; ChIP-Seq
Cells were fixed with 1% formaldehyde (Thermo Scientific) at room temperature for 10 min, followed by quenching with glycine for 5 min at room temperature. The fixed cell pellets were lysed in 1% SDS lysis buffer supplemented with protease inhibitor cocktail tablet (Roche), and sonicated using Bioruptor (Diagenode). The cell lysate was precleared through centrifuging in dilution buffer and incubating with Protein G Dynabeads (Invitrogen) overnight at 4 °C. The precleared lysate was added into the antibody-conjugated beads, and incubated overnight at 4 °C, with rotation. The incubated beads were then washed thrice by 0.1% SDS lysis buffer, once by high salt wash buffer, once by lithium chloride wash and once by the TE buffer. After washing, the incubated beads were added into the elution buffer with RNase A (Qiagen) followed by decrosslinking with Proteinase K (Ambion) at 37 °C overnight. ChIP DNA was extracted back by using the QIAquick PCR purification kit (Qiagen), and quantitated using Qubit High Sensitivity dsDNA Assay (Invitrogen). ChIP-seq library construction was prepared by using ThruPLEX DNA-seq 48D Kit (Rubicon) according to the instruction.