Total RNA was extracted from hESO-14 or PBNT1 cells (passage 5) using the RNeasy Lipid Tissue Mini Kit according to protocol (QIAGEN). mRNA libraries were constructed using the TruSeq Stranded RNA LT Kit (Illumina, San Diego, CA) and 4 µg total RNA according to manufacturer's instructions to confer strand specificity. mRNA was purified using the RNA Purifications Beads per Illumina protocol instructions. The mRNA was incubated in the Elute, Prime, and Fragment Mix at 94˚C for 8 min to fragment mRNA followed by first strand of cDNA and second strand synthesis. The cDNA was ligated to adapter and enriched with PCR to create the final cDNA library per Illumina protocol specifications (15 cycles of PCR). The reaction products were purified using AMPure XP beads according to manufacturer’s specifications. RNA-seq libraries were sequenced using the Illumina HiSeq 2500 (Illumina) instrument per manufacturer’s instructions. Sequencing of libraries was performed with Single Read Cluster Kit v3, 100 cycles (Illumina).
GEO Accession
GSM2091362