Small RNA Library Generation and Sequencing

The quality of total RNA was analyzed with both a Shimadzu 206-97213C BioSpec-nano analyzer system and a denaturing polyacrylamide gel electrophoresis system. A small RNA library was generated according to the Illumina sample preparation instructions (Chen et al., 2012 (link)). The RNA fragments were reverse transcribed using M-MuLV (Invitrogen) with reverse transcription (RT) primers (as recommended by Illumina) to generate single-stranded cDNA. The cDNA was subsequently amplified with Pfx DNA polymerase (Invitrogen) using 20 PCR cycles and the Illumina small RNA primer set. PCR products were purified, and the recovered cDNA was precipitated and quantified with both a NanoDrop spectrophotometer (Thermo Scientific) and a TBS-380 mini-fluorometer (Turner Biosystems) using the PicoGreenH dsDNA quantitation reagent (Invitrogen). The sample concentration was adjusted to 10 nM, and 10-mL final volumes were used for the sequencing reaction. The purified cDNA library was used for cluster generation (on the Illumina Cluster Station). The cDNA was subsequently sequenced on an Illumina HiSeq2000 machine, following the manufacturer's instructions.