GSM1259845: T2h Blood; Staphylococcus epidermidis; RNA-Seq
Total RNA was extracted using RNeasy mini kit (Qiagen) with some alterations in the bacterial cell lyses procedure. Lyses was performed as described in França et al., PLoS One 2012. In brief, biofilm pellets were suspended in 500 µL of in the lyses buffer provided by the kit plus 500 µL of phenol. This supension was then transfered into Lysing Matrix B tubes (MPBiomedicals) and using a FastPrep BioSavan101 (Therno Scientific) the cells were lysed. Finally, the tubes were centrifuged and the suspension transfered into a new tube. An equal volume of 70% of ethanol was added. The suspension was transfered to the RNeasy mini kit columns and following the manufacturer's instructions total RNA was isolated. Thereafter, total RNA was treated with TURBO DNase (Ambion) and 1:5 Acid-phenol:chloroform in order to remove genomic DNA. Additionally total RNA was treated with MICROBEnrichTM (Ambion) and Ribo-ZeroTM rRNA removal kit for Gram-positive bacteria (Epicentre) to remove eukaryotic RNA and bacterial ribosomal RNA, respectively. Libraries were prepared according to Illumina's TruSeq RNA Sample Prep kit version 2. Briefly, 400 ng of previosuly purified mRNA was fragmented and used for the synthesis of double stranded complementary DNA. Thereafter, end-repair, single ‘A’ nucleotide addition to the 3' end of the blunt fragments, adaptor ligation and DNA fragments enrichment was performed to complete libraries construction. Validation of the constructed libraries was assessed through quantitative PCR and Agilent 2200 TapeStation High SensitivityD1K ScreenTapes.
GEO Accession
GSM1259845