title: GSM2027617: E45-2; Capra hircus; RNA-Seq
library_construction_protocol: For RNA-seq, total RNA was extracted from each embryo, 10 mg of total RNA was used for RNA sequencing (RNA-seq) library preparation. Polyadenylated mRNAs were purified and concentrated with Magnetic Beads Oligo (dT) (Invitrogen, Carlsbad, CA, USA) before used for directional RNA-seq library preparation. Purified mRNAs were iron fragmented at 95oC followed by end repair and 5' adaptor ligation. Then, reverse transcription was performed with RT primer harboring 3' adaptor sequence and randomized hexamer. The cDNAs were purified and amplified and PCR products corresponding to 200-500 bps were purified, quantified and applied to Illumine NextSeq 500 system 151 nt pair-end sequencing by ABlife, Inc. (Wuhan, China). Total RNA was extracted by using TRIzol® reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions, and RQ1 DNase (Promega, Madison, WI, USA) was used to remove contaminating genomic DNA. The quality and quantity of the purified RNA was monitored at the ratios of A260/A280 and A260/230 on SmartSpec Plus Spectrophotometer (BioRad, Philadelphia, PA, USA). RNA integrity was further verified by 1.5% agarose gel electrophoresis.