Illumina-based mRNA-seq Library Preparation

Illumina-based mRNA-seq libraries were prepared from 1 μg RNA following previously published methods^{3 (link)}. mRNA was purified from total RNA using Sera-mag oligo(dT) magnetic beads (GE Healthcare Life Sciences) and then fragmented in the presence of divalent cations (Mg²⁺) at 94 °C for 6 min. The resulting fragmented mRNA was used for first-strand cDNA synthesis using random hexamers and reverse transcriptase, followed by second-strand cDNA synthesis using DNA polymerase I and RNaseH. Double-stranded cDNA was end-repaired using T4 DNA polymerase, T4 polynucleotide kinase and Klenow polymerase. The DNA fragments were then adenylated using Klenow exo-polymerase to allow the ligation of Illumina Truseq HT adapters (D501–D508 and D701–D712). All enzymes were purchased from Enzymatics. Following library preparation, quality control and quantification were performed using a 2100 Bioanalyzer instrument (Agilent) and the Quant-iT PicoGreen dsDNA Reagent (Invitrogen), respectively. Libraries were sequenced using Illumina HiSeq4000 sequencers to generate 50-bp single-end reads.