GSM4257609: C; Escherichia coli; RNA-Seq
Total RNA was isolated using RNeasy Mini Kit (Qiagen) using manufacturer's instructions. RNA sequencing libraries were prepared with Illumina-compatible NEBNext® UltraTM Directional RNA Library Prep Kit (New England BioLabs, MA, USA) at Genotypic Technology Pvt. Ltd., Bangalore, India. ~2.5μg of total RNA was taken for rRNA depletion using Ribo-Zero rRNA Removal Kit (Bacteria). ~50 ng of Qubit quantified ribodepleted RNA was taken for fragmentation and priming. The fragmented and primed mRNA 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 PCR magnetic beads (Magbio Genomics Inc, USA). The purified cDNA was end-repaired, adenylated and ligated to Illumina multiplex barcode adapters as per NEBNext® UltraTM Directional RNA Library Prep Kit protocol.
GEO
GSM4257609