title: GSM3182761: E14-ESC-2; Mus musculus; RNA-Seq
library_construction_protocol: Total RNA was extracted by Trizol The cDNA libraries were prepared using the NEXTflex™ Illumina Rapid Directional RNA-Seq Library Prep Kit (BioO Scientific) as per the manufacturer's instructions. To summarize, polyA RNA was purified from 100 ng of total RNA using oligo (dT) beads. The extracted mRNA fraction was subjected to fragmentation, reverse transcription, end repair, 3'– end adenylation, and adaptor ligation, followed by PCR amplification and SPRI bead purification (Beckman Coulter). The unique barcode sequences were incorporated in the adaptors for multiplexed high-throughput sequencing. The final product was assessed for its size distribution and concentration using the BioAnalyzer High Sensitivity DNA Kit (Agilent). Pooled libraries were diluted to 2 nM in EB buffer (Qiagen) and then denatured using the Illumina protocol.