anti-mouse POLR3GL ZCH10079-1434 Ab, raised against peptide RPPKSTDDKEETIQK
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Cell lines : approximately 10 million subconfluent IMR90 or Hepa 1-6 cells were used per ChIP. The protocol used was similar to the one described by (O'Geen et al. 2006). Cells were directly crosslinked in the culture medium for seven minutes with 1% formaldehyde. The chromatin was sonicated to an average size of 200-600 base pairs. Mouse livers : livers were perfused with 5 ml of PBS through the spleen, immediately homogenized in PBS containing 1% formaldehyde, and then processed as described in (Ripperger and Schibler 2006). Aliquots of sonicated chromatin were mixed with different antibodies and incubated overnight at 4˚C on a rotating wheel. Immunoprecipitated material was recovered by addition of 15 µl of protein A agarose beads (pre-blocked with 10 µg/ml of BSA and 10 µg/ml of salmon sperm DNA) and incubation for 1 h at room temperature on a rotating wheel. The beads were washed with dialysis and wash buffer (O’Green et al., 2006). De-crosslinking, RNase A treatment, proteinase K treatment, and DNA purification were performed as described in (O’Green et al., 2006).Ten ng of immunopurified chromatin as well as input DNA was used to prepare sequencing libraries with the ChIP-Seq Sample Preparation Kit (Illumina; San Diego, California, USA; Cat. No IP-102-1001) according to the protocol supplied by the manufacturer