title: GSM5230844: RNA_Pcgf6_WT_EpiLC1; Mus musculus; ssRNA-seq
library_construction_protocol: EpiLC induction was performed essentially as described previously (Hayashi et al., Cell, 2011) with minor modifications. Briefly, pESCs were adapted to 2i (PD0325901, CHIR99021) + LIF feeder-free culture conditions to transit to nESCs. The EpiLCs were induced by plating nESCs in a well coated with human plasma fibronectin (Millipore) in N2B27 medium containing Activin A (R&D), bFGF (Gibco) and KnockOut Serum Replacement (KSR; Gibco). For cKO cells, 4-hydroxytamoxifen (4OHT; Sigma, dissolved in ethanol) at a final concentration of 100 nM was added to culture medium to induce Cre recombinase activity. Total RNA was extracted from 10 K cells by using RNeasy micro kit (Qiagen) with on-column DNaseⅠ treatment. rRNA was depleted by NEBNext rRNA Depletion Kit (NEB) and cDNA was synthesized by NEBNext UltraII RNA First Strand Synthesis Module (NEB) and double-stranded using NEBNext UltraII Directional RNA Second Strand Synthesis Module (NEB). Following purification of double-stranded DNA by AMPure XP beads, library was constructed as described previously with modifications (Brind'Amour et al., Nat Commun, 2015). Briefly, end repair and A-tailing were performed using NEBNext UltraII End Repair/dA-Tailing Module (NEB) and ligation was carried out using NEBNext Ultra II Ligation Module (NEB) with an in-house adaptor which is compatible with Illumina sequencers. After purification of adaptor-ligated products by AMPure XP beads, libraries were amplified with unique indexing primers for 12 cycles. Following purification of the amplified libraries by AMPure XP beads, libraries were pooled, run on 2% E-Gel EX Gel (ThermoFisher Scientific) and the fragments from 200-600 bp was excised and purified by Zymoclean Gel DNA Recovery Kit (Zymo Research).