GSM9687936: Pcgf5_siRNA_RNA-seq_L2C_rep1; Mus musculus; RNA-Seq
RNA-seq library was generated from ten embryos per each group, by using SMART-Seq v4 PLUS Kit (Takara Bio USA) according to the manufacturer's manual. ChIP-seq library were generated from morula-stage embryos at 72 hpi were collected as approximately 40 embryos per biological replicate. ChIP for small cell numbers was performed with a Low Cell ChIP-Seq Kit (Active Motif) according to the manufacturer's manual (version A3) with some modifications. The morulae were freed from the zona pellucida by using pronaseK before crosslinking with formaldehyde. After crosslink-quenching, the sample was sonicated to shear chromatin using a Bioruptor UCD-250 (Cosmo Bio) for 30 x 30 s with 30-s pauses in ice-water. The sample was centrifuged for 2 min at 18,000 x g and the supernatant (200 μL) was transferred to a new tube. The 200-µL sample of sheared chromatin was processed for ChIP using anti-H2AK119ub1 antibody (8240; Cell Signaling Technology). The ChIPed DNA was decrosslinked, purified, and resuspended in 40 µL low-EDTA TE buffer. Sequencing libraries of the ChIPed DNA were constructed by using Next Gen DNA Library Kit and Next Gen Indexing Kit (Active Motif) following the manufacturer's instruction. SMART-Seq v4 PLUS Kit (Takara Bio USA) and Low Cell ChIP-Seq Kit (Active Motif) was used according to the manufacturer's manual.