RNAseq of LMR001 TY control
After 2,5 hours incubation in TY medium, 1/10th volume of stop buffer (ethanol containing phenol at a final concentration of 5%) was added to each culture (6 relicates) to inhibit RNAses and centrifuged 4 minutes at 8,000 g and 4C. The pellets were snap frozen in liquid nitrogen and kept in the freezer at -80C until use.Total RNAs were purified from each replicate with the RiboPureTM kit (AMBION) and included a DNAse I treatment according to manufacturers recommendations. RNA extracts from the 6 replicates were then pooled and subjected to 2 successive rounds of rRNA subtractions with Microbe ExpressTM kit (AMBION) following manufacturers recommendations. Purified mRNAs were sent to Montpellier Genomix for sequencing. The RNAseq library was prepared following illumina kit instructions. Briefly, 100 ng of mRNA was first fragmented chemically, reverse transcribed using random primers and SuperScrit IITM to generate first strand cDNAs. The second strand was synthesized using DNA polymerase I and RNAse H. The cDNA fragments were then end-repaired, a single A base was added and adapters were ligated. These products were enriched by PCR to select for cDNA fragments containing adaptors and to create the final cDNA library. The efficacy of the library construction was checked in a quality control step that involved measuring the adapter-cDNA size and concentration on an Agilent DNA 1000 chip. Sequencing libraries were denatured with sodium hydroxide and diluted to 6 pM in hybridization buffer for loading onto a single lane of an Illumina HiSeq 2000 flowcell V1.5. Cluster formation, primer hybridization and single-read, 100 sequencing cycles were performed on cBot and HiSeq2000 (Illumina, San Diego, CA) respectively.