GSM4907356: ZNF416_FLAG_rep2; Homo sapiens; ChIP-Seq
ZNF416-FLAG expressing IMR90 cells were subjected to ChIP-seq for FLAG as previously described. Briefly, IMR90 cells (n=2 biological replicates) stably expressing ZNF416-FLAG were cross-linked with 1% formaldehyde (Sigma Aldrich, St. Louis, MO, USA) for 10 minutes, followed by quenching with 125 mM glycine (Sigma Aldrich, St. Louis, MO, USA) for 5 minutes at room temperature. Fixed cells were washed with 1X TBS (Thermo Fisher Scientific, Waltham, MA, USA). IMR90 cells were then resuspended in cell lysis buffer (10 mM Tris-HCl, pH7.5, 10 mM NaCl, 0.5% NP-40) and incubated on ice for 10 minutes. The lysates were washed with MNase digestion buffer (20 mM Tris-HCl, pH7.5, 15 mM NaCl, 60 mM KCl, 1 mM CaCl2) and incubated for 20 minutes at 37 °C in the presence of MNase. After adding the same volume of sonication buffer (100 mM Tris-HCl, pH8.1, 20 mM EDTA, 200 mM NaCl, 2% Triton X-100, 0.2% Sodium deoxycholate), the lysate was sonicated for 10 minutes (30 sec-on / 30 sec-off) in a Bioruptor instrument (Diagenode, Denville, NJ, USA) and then centrifuged at 15,000 rpm for 10 minutes. The cleared supernatant equivalent to about 15 million cells was incubated with 30 µl of prewashed anti-FLAG M2 magnetic beads (Sigma, Catalogue #: M8823) (Sigma Aldrich, St. Louis, MO, USA) on a rocker overnight. The beads were extensively washed with ChIP buffer, high salt buffer, LiCl2 buffer, and TE buffer. Bound chromatin was eluted and reverse-crosslinked at 65 °C overnight. DNA was treated with RNase A and Proteinase K (Qiagen, Valencia, CA, USA) and then purified using a Min-Elute PCR purification kit (Qiagen, Valencia, CA, USA). Sequencing libraries were prepared from 5 - 10 ng of ChIP and input DNA with the ThruPLEX® DNA-seq Kit V2 (Rubicon Genomics, Ann Arbor, MI) and were sequenced to 51 base pairs from both ends using an Illumina HiSeq 4000 instrument (Illumina, San Diego, CA, USA).