GSM5288394: SC35 ChIP-Seq WT_CT12C; Mus musculus; ChIP-Seq
ChIP for SC35 was performed using anti-SC35 antibody (ab11826, Abcam). Mouse liver samples were submerged in PBS + 1% formaldehyde, cut into small (~1 mm3) pieces with a razor blade and incubated at room temperature for 15 minutes. Fixation was stopped by the addition of 0.125 M glycine (final concentration). The tissue pieces were then treated with a TissueTearer and finally spun down and washed twice in PBS. Chromatin was isolated by the addition of lysis buffer, followed by disruption with a Dounce homogenizer. The chromatin was enzymatically digested with MNase. Genomic DNA (Input) was prepared by treating aliquots of chromatin with RNase, Proteinase K and heated for reverse-crosslinking, followed by ethanol precipitation. Pellets were resuspended and the resulting DNA was quantified on a NanoDrop spectrophotometer. An aliquot of chromatin (30 μg) was precleared with protein A agarose beads (Invitrogen). Genomic DNA regions of interest were isolated using 4 μg of antibody. Complexes were washed, eluted from the beads with SDS buffer, and subjected to RNase and proteinase K treatment. Crosslinking were reversed by incubation overnight at 65 °C, and ChIP DNA was purified by phenol-chloroform extraction and ethanol precipitation. The DNA libraries were prepared at University of Pittsburgh and sequenced at Gene by Gene, Ltd per standard protocols. DNA libraries were prepared with Ovation® Ultralow V2 DNA-Seq library preparation kit (NuGen) using 1ng input DNA. The size selection for libraries were performed using SPRIselect beads (Beckman Coulter) and purity of the libraries were analyzed using the High Sensitivity DNA chip on Bioanalyzer 2100 (Agilent). The prepared libraries pooled and sequenced using Nova-Seq 6000 (Illumina), generating ~40 million 101 bp single-end reads per samples.