GSM6235702: HEK293_WT_KLF4_IP_3; Homo sapiens; ChIP-Seq
Chromatin was prepared using the truChIP® Chromatin Shearing Kit (PN520154, Covaris) according to the kit procedure. Briefly, 24 hours post-transfection, cells were fixed with formaldehyde and lysed to prepare nuclei. Their chromatin was sheared for 16 minutes/sample in a 1-mL volume using an E220 Evolution sonicator (Covaris) with the instrument settings described in the kit. Five hundred µg of chromatin for each sample was IP-ed with 40 microliters of anti-FLAG® M2 Magnetic Beads (M8823, Sigma-Aldrich) in a rotary holder overnight at 4 oC. The beads were washed once with 1x RIPA Lysis Buffer (20-188, Sigma-Aldrich), followed by four more washes with RIPA Wash buffer (RIPA Lysis buffer with an increased concentration of NaCl to 500 mM). The washed beads were reverse-crosslinked by treating with Proteinase K overnight at 65 oC in a 200-µL volume. Precipitated genomic DNA was purified using a GeneJET PCR Purification Kit (FERK0701, Fisher Scientific). ChIP-seq libraries were prepared according to Swift Bioscience protocols and were sequenced using 150 bp paired-end sequencing on an Illumina NovaSeq, producing an average of 177,095,140 reads per library.