GSM6256971: BulkRNA AFE, replicate A; Homo sapiens; RNA-Seq
Total RNA from hESCs, DE, AFE, and PE cells was isolated by collecting cells using TRI Reagent (Zymo Research), followed by column purification and DNAse treatment using Direct-zol RNA MiniPrep Kit (Zymo Research), and quantification by Nanodrop (Thermo Scientific). 1 ug of RNA from three biological replicates of hESCs, DE, AFE and PE conditions was quantified using Qubit 2.0 Fluorometer (Life Technologies, Carlsbad, CA, USA) and RNA integrity was checked using Agilent TapeStation 4200 (Agilent Technologies, Palo Alto, CA, USA).  RNA sequencing libraries were prepared using the NEBNext Ultra RNA Library Prep Kit for Illumina using manufacturer's instructions (NEB, Ipswich, MA, USA). Briefly, mRNAs were initially enriched with Oligod(T) beads.  Enriched mRNAs were fragmented for 15 minutes at 94 °C. First strand and second strand cDNA were subsequently synthesized. cDNA fragments were end-repaired and adenylated at 3' ends, and universal adapters were ligated to cDNA fragments, followed by index addition and library enrichment by PCR with limited cycles. The sequencing library was validated on the Agilent TapeStation (Agilent Technologies, Palo Alto, CA, USA), and quantified by using Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA) as well as by quantitative PCR (KAPA Biosystems, Wilmington, MA, USA).