title: GSM6133151: HSC-5 p63 IP 2; Homo sapiens; ChIP-Seq
library_construction_protocol: HSC-5 cells were harvested, crosslinked with 1% formaldehyde for 12 min at room temperature, and quenched with 0.125 M Glycine for 10 mins at room temperature. Following 1 PBS wash, 10 x106 cells were lysed in 1 ml Cell Lysis Buffer (10 mM Tris-HCl pH 8.0, 10 mM NaCl, 0.2% NP-40, protease inhibitor) for 10 min on ice. Nuclei were collected by centrifugation at 4,200 rpm for 30 sec, lysed in 500 µl Nuclei Lysis Buffer (50 mM Tris-HCl pH 8.0, 10 mM EDTA, 1% SDS, protease inhibitor) for 15 min on ice, and sonicated using the Bioruptor (Diagenode) for 15 min at 4ºC with the following settings: medium intensity, On/Off 30s, for 15 cycles. The sonicated chromatin was centrifuged at 13,000 rpm for 15 min at 4ºC. 50 µl of the supernatant was stored at 4ºC as Input chromatin DNA. Remaining supernatant was diluted in 3.5 ml IP Dilution Buffer (20 mM Tris-HCl pH 8.0, 2 mM EDTA, 150 mM NaCl, 1% Triton X-100, 0.01% SDS), incubated with p63 antibody for 2 hrs at 4ºC, and then combined with 30 µl of magnetic beads to rotate overnight at 4ºC. The following day, beads were washed once with IP Wash Buffer I (20 mM Tris-HCl pH 8.0, 2 mM EDTA, 50 mM NaCl, 1% Triton X-100, 0.1% SDS), twice with High Salt Buffer (20 mM Tris-HCl pH 8.0, 2 mM EDTA, 500 mM NaCl, 1% Triton X-100, 0.01% SDS), once with IP Wash Buffer II (10 mM Tris-HCl pH 8.0, 1 mM EDTA, 250 mM LiCl, 1% NP-40, 1% sodium deoxycholate), and twice with TE buffer (50 mM Tris-HCl pH 8.0, 10 mM EDTA). Immunoprecipitated chromatin (ChIP DNA) was eluted in 200 µl Elution Buffer (50 mM Tris-HCl pH 8.0, 10 mM EDTA, 1% SDS) by incubation at 65ºC for 15 min. Input DNA was  then reverse-crosslinked along with ChIP DNA using 12 µl of 5 M NaCl and 1 µg/ml RNase A in 65ºC waterbath overnight, followed by treatment with 4 µg/ml Proteinase K at 42ºC for 2 hrs and purification was conducted using MinElute PCR purification kit (QIAGEN). Next-gen sequencing was used to identify enriched chromatin in the IP samples. Data was processed in Galaxy where FastQC was first used to confirm the quality of the samples. Bowtie mapper was then used to map reads to the reference genome and peak calling was performed through MACS14. Data was visualized in the UCSC genome browser. Finally, MEME-ChIP analysis was applied via the MEME suite to confirm enrichment of the p63 binding motif [35]. The experiment was run in duplicate and common targets were evaluated for further study. For p63 IP 2, trimmomatic was used to improve the quality of reads before proceeding with bowtie mapper. Libraries were constructed following the Illumina TruSeq ChIP Protocol