GSM5416444: 293T-ISChIP-DdCBE-WT-rep1; Homo sapiens; ChIP-Seq
Transfected HEK293T cells were harvested and cross-linked with 0.25% formaldehyde on ice for 5 min. After fixation, cells were quenched via adding 2.5 M glycine and incubating on ice for 5 min. The fixed cells were washed twice with 1% BSA/PBS and conjugated with adequate prepared Con-A beads. Next, the cell-bead mixture was incubated with the primary antibody (anti-FLAG, sigma, F1804), the secondary antibody (donkey anti-mouse-Alexa 488, A21202), and purified PAT-MEA/B, step by step. After washing out free PAT-MEA/B, tagmentation was performed at 30°C for 1 h in a thermal cycler and stopped by adding 10 μL 40 mM EDTA. Cells were then lysed at 55°C for at least 3 h in Lysis Buffer. For library preparation, DNA fragments in the same tube were enriched via 10-18 cycles of PCR amplification with Nextera i5 index primer (5'-AATGATACGGCGACCACCGAGATCTACAC[i5]TCGTCGGCAGCGTC-3'), and Nextera i7 index primer (5'-CAAGCAGAAGACGGCATACGAGAT[i7]GTCTCGTGGGCTCGG-3'). After PCR, the library was purified and selected for 200-1,000 bp fragments for sequencing.