GSM7061488: K562 cells, SF3B1 K700E, 4 days, replicate 1 [RNAPIINTDChIPseq_K562_SF3B1K700E_Replicate1]; Homo sapiens; ChIP-Seq
Formaldehyde was directly added to the media to a final concentration of 1% and 1 million cells were incubated for 15 min. Glycine was added to quench the reaction for 5 min. Cells were spun down and pellet was washed twice with PBS .  The cell pellet was lysed on ice in cell lysis buffer, pelleted and resuspended in MNase digestion buffer containing MNase, followed by nuclear lysis in nuclear lysis buffer. Sonication was performed with a sonic Dismembranator.5-10 μg of chromatin was used for each IP. ChIP spike in normalization for the RNAPII ChIP assays was performed using Drosophila-specific chromatin and Drosophila-specific histone variant (H2Av) antibody. 50 ng of Drosophila S2 sheared crosslinked-chromatin was added to 10 μg of chromatin as spike-in control. RNAPII NTD antibody was mixed with diluted chromatin and subjected to IP performed on a rotating wheel at 4°C overnight. Pierce Protein A/G Magnetic beads were added to the chromatin-antibody mixture and 4°C rotation continued for 3 more hours. Beads were washed with wash buffers. Immuno-bound chromatin was eluted at 55°C for 1 hour with elution buffer and de-crosslinked overnight at 65°C. DNA was extracted with phenol:chloroform:isoamyl alcohol and precipitated for 60 min at −20°C with sodium acetate and ethanol. Pellet was washed with 70% ethanol and resuspended in TE buffer. DNA quality and size distribution were checked on Fragment Analyzer. 10 ng of DNA was used for library preparation according to NEBNext® Ultra II DNA Library Prep Kit. Purity and size distribution of the libraries were estimated using Fragment Analyzer.