Figure 1

Flowchart of strand-specific RNA-Seq library preparation. A) Strand-specific sequencing libraries are prepared from total RNA depleted of rRNA by digestion of 5′-P-containing RNA fragments with a 5′-phosphate-dependent exonuclease (Tex) or by enrichment for polyadenylated mRNA. Subsequently, rRNA-depleted RNA is decapped, fragmented, dephosphorylated and re-phosphorylated to obtain RNA fragments with 5′-P and 3′-OH groups. Next, the RNA is ligated to 3′ and 5′-adapters, reverse-transcribed with a primer complementary to the 3′-adapter and the cDNA is PCR amplified for 12-16 cycles using KAPA HiFi polymerase. A recent analysis had compared amplification efficiencies of different polymerases for genomes ranging in GC-content from 67.7% (Boretella pertussis) to 19.3% (P. falciparum) and found KAPA HiFi (Kapabiosystems) to provide the most consistent results for AT-rich genomes [19]. B) Meta-gene coverage plots for libraries prepared from polyA-enriched and Tex-treated RNA. X-axis: scaled gene body, y-axis: scaled coverage.