RNA-Seq of pig conceptus: sample 2 of day 9 of pregnancy
The rRNA was depleted from total RNA using Ribo-Zero rRNA removal Kit. First-strand cDNA was synthesized using ProtoScript II Reverse Transcriptase with random primers and Actinomycin D. The second-strand cDNA was synthesized using Second Strand Synthesis Enzyme Mix. The purified double-stranded cDNA by AxyPrep Mag PCR Clean-up was then treated with End Prep Enzyme Mix to repair both ends and add a dA-tailing in one reaction, followed by a T-A ligation to add adaptors to both ends. Size selection of Adaptor-ligated DNA was then performed using AxyPrep Mag PCR Clean-up, and fragments of ~360 bp were recovered. The dUTP-marked second strand was digested with Uracil-Specific Excision Reagent enzyme. Each sample was then amplified by PCR for 11 cycles using P5 and P7 primers, with both primers carrying sequences which can anneal with flow cell to perform bridge PCR and P7 primer carrying a six-base index allowing for multiplexing.