GSM5021091: 16HBE14o- KLF5 ChIP-seq rep2; Homo sapiens; ChIP-Seq
Cells were lysed in lysis buffer (5mM PIPES pH 8.0, 85mM KCL, 0.5% NP-40, 1x Protease Inhibitor Cocktail (Roche)). Nuclei were isolated by centrifugation, lysed in RIPA buffer (1x PBS, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, Protease Inhibitor Cocktail), and sonicated. Protein-DNA complexes were isolated with rabbit anti-KLF5 (Yang Y, et al. Cancer Biol Ther. 2005) [Calu-3 KLF5 Rep1], Santa Cruz sc-398470 [Calu-3 KLF5 Rep2, 16HBE14o- KLF5], Santa Cruz sc-7962 [Calu-3 CEBPB] DNA ends were blunted using T4 DNA polymerase, Klenow DNA polymerase, and T4 polynucleotide kinase. Next, DNA was incubated with Klenow exo- to add 5' adenine overhangs. Multiplex adaptors were then ligated to the ends and converted to dsDNA using 5 cycles of PCR. DNA was size-selected to contain fragments between 200 and 300bp and PCR amplified for 5 cycles. DNA was purified with 1.2x AMPure XP beads. Sequencing was performed on an Illumina Hi-Seq.