GSM1369855: LYS2_RNA-seq; Clostridioides difficile; RNA-Seq
Bacterial cells were harvested by centrifugation, resuspended in TRIzol reagent (Life Technologies, Cat. No. 15596-026) and mechanically lysed with glass beads on Fast Prep  aparatus. Total RNA from TRIzol was extracted with chloroform and precipitated with isopropanol. 4 μg of total RNA were treated with Ribo-Zero™ rRNA Removal Kit for Gram-Positive Bacteria (Epicentre Biotechnologies). Residual RNA was cleaned up using RiboMinus™ Concentration Module columns (Invitrogen) and eluted directly in the Elute/Frag/Prime buffer of the Illumina TruSeq RNA Sample Preparation Kit v2. The remaining of the protocol has been performed as per the manufacturer's recommendation, except that cDNA was sheared on a Covaris instrument. Libraries were quantified using the Quant-iT™ PicoGreen® dsDNA Assay Kit (Life Technologies) and the Kapa Illumina GA with Revised Primers-SYBR Fast Universal kit (D-Mark). Average size fragment was determined using a 2100 Bioanalyzer (Agilent Technologies) instrument. cDNA libraries were multiplexed on a single sequencing lane and sequenced on a HiSeq2000 system using 9 pM as estimated by qPCR (Kapa Biosystems) for 50 cycles yielding single-end 50 nucleotide reads. Fastq files were generated using HCS v1.5 and BclToFastq.
GEO Accession
GSM1369855