GSM7060124: RNAseq_ESC_BAZ2A_wt_rep2; Mus musculus; RNA-Seq
Total RNA was purified with TRIzol reagent (Life Technologies). The quality of the isolated RNA was determined with a Qubit® 4 Fluorometer and 4200 Tape Station system. Only those samples with a 260 nm/280 nm ratio between 1.8–2.1 and a 28S/18S ratio within 1.5-2 were further processed. The TruSeq Stranded mRNA (Illumina) was used in the succeeding steps. Briefly, total RNA samples (100-1000 ng) were polyA enriched and then reverse-transcribed into double-stranded cDNA. The cDNA samples were fragmented, end-repaired and adenylated before ligation of TruSeq adapters containing unique dual indices (UDI) for multiplexing. Fragments containing TruSeq adapters on both ends were selectively enriched with PCR. The quality and quantity of the enriched libraries were validated using Qubit® 4 Fluorometer. The product is a smear with an average fragment size of approximately 260 bp. Libraries were normalized to 10 nM in Tris-Cl 10 mM, pH8.5 with 0.1% Tween 20. The Novaseq6000 (Illumina) was used for cluster generation and sequencing according to standard protocol. Sequencing was single end at 100 bp.