Total RNA
RNA sequencing libraries were prepared with Illumina-compatible NEBNext  Ultra  Directional RNA Library Prep Kit (New England BioLabs, MA, USA) at Genotypic Technology Pvt. Ltd., Bangalore, India. 70 ng of RNA was taken for denaturation and random hexamer priming. Primed RNA was further subjected to first strand synthesis in the presence of Actinomycin D (Gibco, life technologies, CA, USA) followed by second strand synthesis. The double stranded cDNA was purified using HighPrep magnetic beads (Magbio Genomics Inc, USA). Purified cDNA was end-repaired, adenylated and ligated to Illumina multiplex barcode adapters as per NEBNext  Ultra  Directional RNA Library Prep Kit protocol.Adapter ligated cDNA was purified using HighPrep beads and was subjected to 15 cycles of Indexing-PCR (37  C for 15mins followed by denaturation at 98 C for 30 sec, cycling (98 C for 10sec, 65 C for 75sec) and 65 C for 5mins) to enrich the adapter-ligated fragments. Final PCR product (sequencing library) was purified with HighPrep beads, followed by library quality control check. Illumina-compatible sequencing library was quantified by Qubit fluorometer (Thermo Fisher Scientific, MA, USA) and its fragment size distribution was analyzed on Agilent 2200 Tapestation.