GSM5342984: IP_ChIP-seq_RNAPII_siCMTR1_2; Mus musculus; ChIP-Seq
Cells were cross-linked by 1% formaldehyde for 10 min. Then the glycine was added to quench the reaction. The cell lysates were prepared as described (Varshney et al., 2018).The chromatin was sonicated in Bioruptor (Diagenode) at high power for 20 min (30 s on/off, 20 cycles). 25-50 micrograms of the sonicated chromatin were used for each IP. 50μl (10%) from each diluted extract for IP were taken as the inputs. For ChIP using anti-HA beads, 20μL of anti-HA magnetic beads (Pierce) were added to lysates from CMTR1/RNMT KI cells (as IPs) and E14 cells (as control) for 4°C overnight incubation. For RNAP II ChIP, 2μg of anti-Rpb1 (CST 14958) were added to lysates from E14 cells for 4 °C overnight reaction before the incubation with protein G Dynabeads (Life Technologies) for additional 2 hours. The beads were washed and eluted as described (Varshney et al., 2018) before the proteinase K treatment and reverse crosslinking at 42 °C overnight. The eluted DNA was purified by the QIAquick PCR purification kit (QIAGEN). Libraries were prepared using NEBNext® Ultra™ II DNA Library Prep Kit and NEBNext® Multiplex Oligos (Index Primers Set 1) for Illumina.
GEO
GSM5342984