DF1 cells
RNA sequencing libraries were prepared with Illumina-compatible NEBNext® Ultra™ Directional RNA Library Prep Kit at Genotypic Technology Pvt. Ltd., Bangalore,India. 1µg of total RNA was taken for mRNA isolation, fragmentation and priming. Fragmented and primed mRNA was further subjected to first strand synthesis in the presence of Actinomycin D followed by second strand synthesis. The double stranded cDNA was purified using JetSeq magnetic beads (Bio, # 68031). 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 JetSeq 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 JetSeq beads, followed by library quality control check.