GSM5016609: M5a1 WT Nfix rep2; Klebsiella oxytoca; RNA-Seq
Cell cultures were fixed with a 1/10 volume of 5% phenol/95% ethanol (v/v), harvested by centrifugation at 4000 x g and stored at -80°C. Cell pellet samples were shipped on ice to Vertis Biotechnologie AG (Freising, Germany) for RNA extraction and sample processing.Total RNA was isolated via lysozyme lysis and the mirVana microRNA isolation kit (Ambion). DNase was used to specifically remove contaminant DNA and ribosomal RNA was depleted using the Ribo-Zero rRNA Removal Kit for bacteria (Illumina). The primary RNA transcripts were fragmented using ultrasound (4 pulses of 30 s each at 4°C) prior to ligation of 3' oligonucleotide adapters. First-strand cDNA synthesis via Moloney Murine Leukemia Virus (M-MLV) reverse transcriptase was primed using the 3' adapter. Following purification of first-strand cDNA, a 5' Illumina TruSeq sequencing adapter (Illumina), inclusive of an 8 nt barcode sequence, was ligated to the 3' end of the antisense cDNA prior to PCR-amplification of double-stranded cDNA to about 10-20 ng/μl (12-13 thermocycles) using a high fidelity DNA polymerase. cDNA was purified, pooled in equimolar quantities and size-fractionated (200-500 bp) using the Agencourt AMPure XP kit (Beckman Coulter Genomics) prior to analysis by capillary electrophoresis. cDNA pools were quality checked on a Shimadzu MultiNA microchip electrophoresis system prior to strand-specific sequencing on an Illumina NextSeq 500 system, utilising a read length of 75 nt and a depth of 100 million reads.