Illumina HiSeq 4000 paired end sequencing; ChIP-Seq of Dazl in mouse embryonic stem cells
ChIP-Seq of Dazl in mouse embryonic stem cells
The ChIP procedure and analysis were carried out essentially as described previously (PMID: 27773674).  Briefly, mESCs were grown in 2iL medium. The cells were detached, and fixed by 1.5% formaldehyde in PBS for 15min. Chromatin was solubilized by sonication, and sheared to < 500 bp fragments. Dazl immunoprecipitation was carried out using CST antibodies #8042 overnight at 4 Â°C. Following washing steps, chromatin was eluted, and DNA was purified The eluted chromatin was treated with proteinase K (20ug) to degrade proteins. Then the samples were heated at 95 C for 10 min to reverse the crosslinking. Then DNA was purified by SPRI magnetic beads 2x vol of the samples. After washing with 80% ethanol, DNA was eluted in Tris HCl 10mM. DNA library was prepared using NEBNext Illumina Ultra II components. The libraries were indexed using NEBNext Multiplex Oligos. The size distribution of the libraries were checked using TapeStation, and the libraries were pooled.