GSM2350363: ChIP-Seq input in JHU-029 HNSCC; Homo sapiens; ChIP-Seq
JHU029 cells were subjected to additional cross-linking protein-protein interaction with 2 mM EGS followed by cross-linking with 1% (v/v) formaldehyde. Cells were then lysed in RIPA buffer  for 1-2hr at 40C. Nuclear lysates were sonicated and the sonicated chromatin was precleared with ProteinG sepharose beads (GE healthcare) pre-blocked with BSA and sonicated salmon sperm DNA. Samples were incubated with 2 μg of p63 antibody (H129, Santacruz) for overnight at 40C and with beads for additional 2 hrs. Beads were washed in wash buffer. After the wash, beads were incubated for 3 hrs at 550C, then overnight at 650C in TE buffer containing SDS, RNase A, Proteinase K. DNA was purified via Qiaquick PCR purification kit (28106, Qiagen) as per manufacturer’s instructions. Percentage of input was calculated by using 10% as standard. Libraries were prepared for ChIP-sequencing using Illumina kit (cat number, 1005709, 1003473).