Cappable-seq Library Generation from RNA

RNA samples from triplicate normoxic and hypoxic cultures were subjected to enrichment for primary transcripts as previously described [21 (link)], with minor modifications. First, 1.8× Agencourt AMPure XP beads (Beckman) were used to purify RNA in all procedures. Second, to obtain enough primary transcripts for subsequent library construction, enrichment procedure using hydrophilic streptavidin magnetic beads (NEB) was performed for one time. The NEBNext Small RNA Library Prep Set for Illumina (NEB) was used to generate Cappable-seq libraries. To reduce the concentration of adaptor dimer, the 3′ SR adaptor and 5′ SR adaptor were both used at 4-fold dilutions. After 21 cycles of PCR amplification, the libraries were purified using a QIAquick PCR Purification Kit (QIAGEN) and 1.5× Agencourt AMPure XP beads (Beckman). The concentration and size distribution of the libraries were determined by Qubit 3 (Invitrogen) and Bioanalyzer DNA 1000 (Agilent) respectively. Sequencing was performed by the Illumina HiSeq X Ten platform.