GSM4656177: RNA-Seq_Caulobacter_crescentus_NA1000(flaF)_rep1; Caulobacter vibrioides; RNA-Seq
Cells were lysed in a Ready-Lyse lysozyme solution (Epicentre Technologies) according to manufacturer's instructions and lysates were homogenized through QiaShredder columns (Qiagen). Total RNA was extracted using the RNeasy® extraction kit (Qiagen). Briefly, RNA was then purified on RNeasy kit column. To remove DNA from samples, a first treatment using the On-column DNase I digestion kit (Qiagen) was performed and RNA was extracted according to manufacturer's instructions. Extracted RNA was subjected to a second DNase treatment using Promega RQ1 at 1 unit/µg RNA and then purified by phenol/chloroform/isoamyl (Sigma) extraction and ethanol precipitation. Purified total RNA was submitted to the iGE3 Genomics Platform in the CMU (University of Geneva, Faculty of Medicine) for depletion of ribosomal RNA, library preparation, next generation sequencing and differential expression analysis. The total RNA subjected to library preparation using TruSeq ribodepleted Stranded mRNA (SR100-Illumina Hiseq4000) according to manufacturer's protocols. The sequencing quality control of the reads was done with FastQC v.0.11.5. For the analysis, the reads were mapped with BWA aligner v.0.7.15 to the NCBI C. crescentus NA1000 reference. The table of counts with the number of reads mapping to each gene feature of the NCBI C. crescentus NA1000 was prepared with HTSeq v0.6p1 (htseq-count). For each experimental series, the differential expression analysis was performed with the statistical analysis R/Bioconductor package edgeR v.3.18.1.