Chryseobacterium sp. (24 hrs) Uranium treated sample replicate 2
RNA sequencing libraries were prepared with Illumina-compatible NEBNext Ultra II Directional RNA Library Prep Kit at Genotypic Technology Pvt. Ltd., Bangalore, India. 200ng-100ng of Qubit-quantified total RNA was taken for rRNA removal using the NEBNext rRNA Depletion Kit (BACTERIA). Ribodepletion was performed by following manufacturers instructions. Briefly, hybridization of RNA to biotinylated rRNA-specific probes (DNA- based) was carried out at 95degC 2 minutes, followed by ramping down the complexes to 22degC, and then followed by RNAse H and DNase I digestion. Further the samples were subjected to 1.8X cleanup and eluted in 7ul NFW. The rRNA-free and cleaned-up RNA was quantified on Qubit fluorimeter. The enriched RNA was subjected to fragmentation, first strand synthesis followed by second strand synthesis. The double stranded cDNA was purified with NEBNext purification beads. The cDNA was end-repaired, adenylated and ligated to Illumina multiplex barcode adapters as per NEBNext Ultra II Directional RNA Library Prep protocol followed by second strand excision using USER enzyme at 37 C for 15mins.