Total RNA Extraction and Library Preparation for RNA-Seq and sRNA-Seq

Isolation of total RNA from various tissues of different rice lines was performed using TRIzol Reagent (Ambion) as per manufacturer’s instructions. The concentration of RNA and purity of the samples were estimated using a Nanodrop Spectrophotometer (Thermo Fisher Scientific) and Qubit Fluorometer (Thermo Fisher Scientific), respectively. An aliquot of the samples was also separated on an Agilent RNA Bio-analyzer chip to check for integrity. RNA sequencing libraries were constructed according to the NEXT-flex Rapid directional mRNA-Seq bundle library protocol at Genotypic Technologies, India, and sequenced on an Illumina NextSeq500 platform. sRNA sequencing libraries were prepared with a TruSeq Small RNA Sample Preparation Guide (Illumina, USA) at Genotypic Technologies, India, and sequenced on an Illumina NextSeq500 platform.