GSM1518909: Biofilm treated with carbapenems for 2 hours rep2; Klebsiella pneumoniae; RNA-Seq
Biofilm wells were rinsed with LB to remove unattached K. pneumoniae cells and Following 2 or 24 hours of treatment, biofilms were removed from the plates and preserved in RNA protect (QIAgen, Valencia, CA, USA).  RNA was extracted using the RNeasy Mini Kit (QIAgen) according to manufacturer’s instructions.  Samples were then treated twice with TURBO DNase (Ambion, Grand Island, NY, USA) according to manufacturer’s instructions to remove contaminating genomic DNA.  RNA was quantified using a NanoDrop ND-1000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA) and analyzed for quality using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). 2-5ug total RNA was used for strand-specific RNA Sequencing (RNASeq) library preparation. rRNA was depleted from total RNA using the Ribo-Zero Magnetic Kit (Epicentre, Madison, WI, USA).  The directional RNAseq library was developed using the NEXTflex Directional RNA-seq (dUTP based) kit (Bioo Scientific, Austin, TX, USA).  Following purification, mRNA was fragmented into small pieces using divalent cations under elevated temperature. The cleaved RNA fragments were copied into first strand cDNA using reverse transcriptase and random primers followed by second strand cDNA synthesis using DNA Polymerase I and RNase H. These cDNA fragments went through an end repair process, the addition of a single ‘A’ base, and ligation of the adapters. The products were further purified and enriched with PCR to create the final RNASeq library. Directionality is retained by adding dUTP during the second strand synthesis step and subsequent cleavage of the uridine-containing strand using Uracil DNA Glycosylase. RNASeq libraries were subjected to quantification process, pooled for cBot amplification and subsequent sequencing run with HiSeq 2000 platform (Illumina, San Diego, CA, USA). After the sequencing run, demultiplexing with CASAVA was employed to generate a FASTQ file for each sample.
GEO
GSM1518909