RNA-seq of Equus caballus: term chorioallantois, pregnant horn
Paramagnetic beads coupled with oligo d(T)25 were combined with total RNA to isolate poly(A)+ transcripts based on NEBNext Poly(A) mRNA Magnetic Isolation Module manual (New England BioLabs Inc.; Ipswich, MA, USA). Prior to first strand synthesis, samples were randomly primed (5  d(N6) 3  [N=A,C,G,T]) and fragmented based on the manufacturers recommendations. The first strand was synthesized with the Protoscript II Reverse Transcriptase with a longer extension period, approximately 30 minutes at 42C. All remaining steps for library construction were used according to the NEBNext UltraTM II Non-Directional RNA Library Prep Kit for Illumina (New England BioLabs Inc.). Final libraries quantity was assessed by Qubit 2.0 (ThermoFisher) and quality was assessed by TapeStation HSD1000 ScreenTape (Agilent Technologies Inc.). The final library size was about 430bp with an insert size of about 300bp and sequenced on an Illumina NovaSeq 6000 system (Illumina; San Diego, CA, USA) with a read length configuration of 150bp, stranded and paired-end.