RNA-Seq and Small RNA Library Preparation

RNA-Seq libraries were prepared according to the protocol described in Zhong et al. (2011) (link). Briefly, mRNA was enriched by magnetic beads with Oligo (dT), and then sheered into short fragments in the fragmentation buffer. First strand cDNA was synthesized using SuperScriptIII reverse transcriptase (Invitrogen) in the presence of dNTPs. Second strand cDNA was synthesized using RNase H (NEB) and the Klenow fragment of DNA polymerase I (NEB) with a dUTP mix at 16 °C for 2.5 h using dUTPs. After end repair, adapters were ligated to the double stranded cDNA, and the dUTP-containing strand was degraded using a uracil DNA glycosylase. sRNA libraries were constructed following the protocol of Chen et al. (2012) (link). Briefly, 10 ug of sRNA was fixed with 3’ and 5’ adapters via ligation. sRNAs were then reverse transcribed using SuperScript III reverse transcriptase, PCR enriched, separated on a 2% agarose gel, and gel purified to enrich fragments of the correct length. Both RNA-Seq and sRNA libraries were sequenced on an Illumina HiSeq 2500 system. Raw RNA-Seq and sRNA reads have been deposited in NCBI SRA under the accession number PRJNA649319.