Clinical E. coli Strain- EC555 (2)
RNA sequencing libraries were prepared with Illumina-compatible NEBNext Ultra II Directional RNA Library Prep Kit (New England BioLabs, MA, USA) at Genotypic Technology Pvt. Ltd., Bangalore, India.  1.5 g of total RNA was taken for rRNA depletion using Ribo-Zero rRNA Removal Kit (Bacteria). ~20-30  ng of Qubit quantified ribodepleted RNA was taken for fragmentation and priming. The fragmented and primed mRNA was further subjected to first strand synthesis followed by second strand synthesis. The double stranded cDNA was purified HighPrep Beads (Magbio, Cat # AC-60050). Purified 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. The adapters used in the study were Illumina Universal Adapter:5-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-3 and Index Adapter: 5-GATCGGAAGAGCACACGTCTGAACTCCAGTCAC [INDEX] ATCTCGTATGCCGTCTTCTGCTTG-3. [INDEX]  Unique sequence to identify sample-specific sequencing data Adapter ligated cDNA was purified using HighPrep Beads and was subjected to 11 cycles for Indexing- (98C for 30 sec, cycling (98C for 10sec, 65C for 75sec) and 65C for 5min) 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 2100 Bioanalyzer.