Small RNA sequencing pigeonpea grown under elevated CO2 and drought
Small RNA libraries for sequencing were constructed at the Genotypic Technology’s Genomics facility according to the Illumina TruSeq Small RNA library protocol outlined in “TruSeq Small RNA Sample Preparation Guide” (Part # 15004197; Rev. F; February 2014). 1g oftotal RNA (60% root; 40% leaf)was used as the starting material. Briefly, 3’ adaptors were ligated to the specific 3’OH group of micro RNAs followed by 5’ adaptor ligation. The ligated products were reverse transcribed with Superscript III Reverse transcriptase by priming with Reverse transcriptase primers. The cDNA was enriched and barcoded by PCR (15 cycles. The library was size selected using PAGE gel in the range of 140 – 160 bp followed by overnight gel elution and salt precipitation using Glycogen, 3M Sodium Acetate and Absolute Ethanol. The precipitate was re-suspended in Nuclease free water. The prepared library was quantified using QubitFluorometer, and validated for quality by running an aliquot on High Sensitivity Bioanalyzer Chip (Agilent).