Strand-specific RNA-seq libraries were prepared in accordance with the protocol described by Waldbauer and coworkers (52 (link)). Briefly, The DNase-treated RNA (100 ng) was fragmented by divalent-cation hydrolysis (Fragmentation Buffer; Ambion, Austin, TX) at 70°C for 12 min to yield fragment sizes between 50 and 300 nt. After precipitation with ethanol, fragments were subjected to poly(A) tailing and end repairing (NEB Reagents). RNA was treated with Antarctic phosphatase (New England Biolabs) and then phosphorylated at the 5′ RNA end with T4 polynucleotide kinase. The transcribing strand was labeled by ligation of a 5′ hybrid DNA-RNA primer and after purification with RNA clean XP beads (Beckman Coulter Genomics), the first-strand cDNA synthesis reaction was carried out with SuperScript II reverse transcriptase (Invitrogen) and Illumina’s poly(T) primer and deoxynucleoside triphosphates (20 mM). Reaction components were removed with Agencourt AMPure XP SPRI beads (Beckman), and primary transcripts were enriched with Pfu Hi Fidelity polymerase (Invitrogen) and the Illumina spacers as primers. Illumina adaptors and bar codes were ligated by PCR in accordance with the manufacturer’s instructions. Libraries were purified with SPRI beads (Beckman) and quantified with a NanoDrop (Bio-Rad). Sequencing was performed with the Illumina technology at Ambry Genetics.