Strand-specific RNA-seq library preparation

Total RNA extracted using the MagJET RNA kit (Thermo Scientific) was first checked for integrity on an Agilent Bioanalyzer 2100; samples with RNA integrity number (RIN) >9.0 were used for subsequent processing. Total RNA was subjected to two rounds of poly(A) selection using oligo-d(T)₂₅ magnetic beads (New England Biolabs). A single-read cDNA library was prepared following the Illumina TrueSeq small RNA protocol for strand-specific RNA-seq with minor modifications (Hoque et al. 2013 (link)). Briefly, poly(A)⁺ RNA was fragmented in an alkaline buffer (NaHCO₃ at pH 9.3) for 2 min at 94°C followed by dephosphorylation with recombinant shrimp alkaline phosphatase (New England Biolabs) and then phosphorylation with T4 polynucleotide kinase (New England Biolabs). After addition of 3′ adapter (5′ adenylated) and 5′ adapter using truncated T4 RNA ligase II (New England Biolabs) and T4 RNA ligase I (New England Biolabs), respectively, RNA was reverse-transcribed using 3′ adapter-specific primer. cDNA was then amplified by PCR for 15 cycles with a universal forward primer and a reverse primer with bar code. The cDNA libraries were purified from an 8% polyacrylamide gel and quantified on an Agilent Bioanalyzer.