GSM2875543: RNAseq_cKO_1; Mus musculus; RNA-Seq
RNA-seq: total RNA was extracted from 500 pachytene cells using RNAqueous-Micro Total RNA Isolation Kit (ThermoFisher, AM1931) and eluted in 10 µl of elution solution. ChIP-seq: wildtype (3268 cells) and Setdb1 cKO (5203 cells) pachytene cells were used for library prep described below. RNA-seq: cDNA was amplified from 1 µl of total RNA by using Smart-seq2 protocol (S. Picelli et al., Nat Methods. 10, 1096-8. 2013; 15 amplification cycles). 10 ng of cDNA was sheared using Covaris E220. Libraries of ~300 bp were generated using Ovation Ultralow DR kit (NuGEN, 0330) with 14 PCR cycles. ChIP-seq: Ultra-low-input native ChIP-seq library was prepared by following a published protocol (J. Brind'Amour et al., Nat. Commun. 6, 6033. 2015). 5% of the samples after micrococcal nuclease digestion were taken as input. 6.25 µl of diluted H3K9me3 antibody (1:100; Active Motif, 39161) per reaction was used for ChIP. NEBNext Adapter for Illumina was used as adapters and adapter-ligated DNAs were amplified by 13 PCR cycles using NEBNext Index primers (New England Biolabs, included in E7335 and E7500). The libraries were size-selected (200-500 bp) using E-Gel (ThermoFisher).