title: GSM3889325: State 1 (Nanog high, Sox2 high) Replicate B RNA-Seq; Mus musculus; RNA-Seq
library_construction_protocol: Cell states were separated by flow cytometric sorting, followed by total RNA isolation (Trizol), DNAse treatment, and quality assessment (BioAnalyzer) rRNA-depleted RNA-sequencing:  Libraries were prepared according to manufacturer's instructions (KAPA HyperPrep with RiboErase HMR, kit KK8561).  In brief, samples were treated with capture oligos followed by rRNA depletion, DNase digestion, RNA fragmented, followed by 1st Strand synthesis and 2nd Strand Synthesis with dUTP incorporation, adapter ligation, and 11 cycles of PCR amplification including barcoding.  The final libraries were QC checked by electrophoresis and qPCR for colony forming units prior to pooling and loading on an Illumina FlowCell (NextSeq 500, 150 bp PE reads).  small RNA (miRNA) sequencing:  Libraries were prepared according to the manufacturer's instructions (NEB small RNA sequencing kit, E7300S).  In brief, samples were treated by 3' adaptor ligation, primer hybridization, 5' adaptor ligation, cDNA synthesis, 13 cycles of pcr amplification followed by library cleanup and QC assessment as above prior to pooling and loading on an Illumina FlowCell (HiSeq2000 1 lane, 40 bp SE reads).