RNA-seq of sus scrofa: mesometrium of pregnant day 12 of replicate 2
After confirming no DNA contamination by agarose gel electrophoresis, RNA integrity was checked using the Agilent 2100 bioanalyzer (Agilent Technologies, USA). The strand-specific library was constructed by Novogene Co., Ltd. (China) with the NEBNext UltraTM Directional RNA Library Prep Kit (NEB, USA). Specifically, rRNA was removed using Epicentre Ribozero rRNA Removal Kit (Epicenter, USA) following the manufacturers protocol. The RNA was fragmented into 250-300bp as a template to synthesize the first strand of cDNA. The RNA was degraded with RNase H, and the second strand of cDNA was synthesized with dNTPs (dUTP, dATP, dGTP, and dCTP) using DNA polymerase I. AMPure XP beads (A63881, Beckman, USA) were then used to screen cDNAs of 350-400 bp after DNA purification, end repair, A-tailing, and sequencing adaptor ligation. cDNA containing dUTP was degraded using USER enzyme. Eventually, the library was constructed through PCR amplification.Adjusting the concentration of the constructed library to 1 ng/ul, and the insert size was confirmed to expected distribution of 250-300 bp with an Agilent 2100 bioanalyzer (Agilent Technologies, USA). The exact concentration of the cDNA library was checked again with qPCR, and the libraries were sequenced on the Illumina PE150 according to data output requirements pooling. Sequencing was performed in a paired-end configuration to generate 2  150 bp reads.