GSM5001935: Global RNA-seq; Flavobacterium psychrophilum; RNA-Seq
Total RNA extraction was performed using hot phenol method. Bacterial cells were collected after addition of ½ volume of frozen killing buffer (20 mM Tris-HCl pH 7.5, 5 mM MgCl2, 20 mM NaN3) to the culture. Cell pellets were frozen in liquid nitrogen and stored at −80 °C. For extraction, the pellets were resuspended into lysis buffer (4M guanidine thiocyanate, 25 mM sodium acetate pH 5.2, 5 g L−1 N-laurylsarcosinate), immediately mixed to hot acid phenol v:v and incubated during 10 min at 65 °C. The aqueous phases were recovered by centrifugation after addition of 400 µL chloroform. The samples were extracted with acid phenol:chloroform:IAA (25:24:1, pH 4.5) and then chloroform. Total RNAs were ethanol precipitated and the pellets resuspended in RNase-free water. Fifty µg of RNA extracts were treated using TurboDNase I (Ambion) to remove residual genomic DNA, then purified using the RNA Clean-Up and Concentration Kit (Norgen Biotek). RNA concentration of the DNase-treated sample was measured using a NanoDrop 1000 Spectrophotometer (NanoDrop Technologies, Inc.) and RNA quality was assessed by capillary electrophoresis using RNA Nano chips with a Bioanalyzer Agilent 2100 (Agilent Technologies, Palo Alto, USA). The 18-condition RNA pool was obtained by mixing each of the extracts in equal quantity. 20 µg of the 18-condition RNA pool were processed using MICROBExpress (Ambion) according to manufacturer's instructions to remove ribosomal RNAs. Efficient rRNA depletion was confirmed by Bioanalyzer 2100. Strand specific transcriptome RNA-Seq library was performed using the TruSeq SBS Kit v3 with no modification.