raw RNA-Seq of Pisum sativum
Small RNA sequencing (SmRNA) library was prepared with TruSeq Small RNA Sample Preparation protocol (Illumina,San Diego, California, U.S.A.). 1000 ng of total RNA was used as starting material. 3 adapters were ligated to the specific 3OH group of micro RNAs followed by ligation of 5 adapter. Adapter ligated fragments were reverse transcribed with Superscript III reverse transcriptase (Invitrogen) by priming with reverse transcription primers. cDNA thus formed was enriched and barcoded by PCR amplification (15 cycles). The amplified library was size selected using Polyacrylamide gel. The library was size selected in the range of 140 bp - 160 bp followed by overnight gel elution and precipitation in presence of glycogen, 3M sodium acetate (Sigma, Saint Louis, Missouri, USA), and absolute ethanol. Pellet was re-suspended in Nuclease-free water (Invitrogen, Whitefield, Bangalore).Illumina-compatible sequencing library was quantified by Qubit fluorometer (Thermo Fisher Scientific, MA, USA) and its fragment size distribution was analyzed on Agilent 2100 Bioanalyzer