Illumina HiSeq 2500 sequencing; ChIP-seq experiment to determine the genomic binding sites of mutant V5-tagged ZFHX2 R1913K protein in a stable-expressing human SH-SY5Y neuroblastoma cell line
ChIP-seq experiment to determine the genomic binding sites of mutant V5-tagged ZFHX2 R1913K protein in a stable-expressing human SH-SY5Y neuroblastoma cell line
A stable transfected SH-SY5Y human neuroblastoma cell line expressing Zfhx2mut at a high level was cultivated in 150mm petri dishes to 75-80% confluency. Cells were cross-linked with freshly prepared 37% formaldehyde to a final 1% concentration at RT on a slow rotating platform for 10 min and crosslinking was terminated by addition of ice cold glycine to a final concentration of 0.125 M for 5 min. Cells were harvested, washed in PBS and stored at -80oC. The chromatin from crosslinked cells was isolated and sheared to ~300 bp of average size fragments (six pulses of 10 min each at High setting on Bioruptor, Diagenode). Each sample had two technical replicas. A single ChIP reaction used chromatin from 7.5x107 cells and either anti-V5 (Abcam, ab15828, 0.7mg/ml) or anti-TBP for positive control (Abcam, ab28175, 1mg/ml) antibodies, incubated overnight at 4oC, then 2 h with M280 Sheep anti-Rabbit IgG Dynabeads (Invitrogen) at RT typically using 5 μg of Ab and 25μl of Dynal beads per 1x106 of cells. Negative control ChIP reactions used rabbit IgG (Abcam). Beads were washed 10 times in 450 μl volume of RIPA ChIP buffer and twice with 200 μl TE buffer with aid of the Precipitor (Abnova). DNA on washed beads was eluted and cross-link reversal was performed in one stage. DNA was purified with IPure kit (Diagenode) and Precipitor (Abnova). Final DNA samples were solubilised in 100 μl with concentration range between 7.5 to 14 ng/μl. DNA was purified with IPure kit (Diagenode) and Precipitor (Abnova). Library preparation was performed on 1 ng of DNA using the MicroPLEX v2 (12 indices) protocol.
Experimental Factor: immunoprecipitate
anti-TBP