GSM3446282: DAP-R Sample_1-C (mRNA); Staphylococcus aureus; RNA-Seq
RNAs were extracted from the samples after 12h of incubation using the NucleoSpin RNA (Macherey-Nagel, Dueren, Germany) kit following manufacturer's protocol with minor modifications. Bacterial cell pellets were lysed by bead-beating procedure in the presence of 50 μL Buffer RA1. Then 3.5 μL ß-mercaptoethanol were added and the lysate was filtrate through NucleoSpin Filters (violet rings). RNA binding condition was adjusted by adding 350 μL of ethanol (70%) to the lysate and the RNA was then extracted with TRIzol reagent (Invitrogen) according to the manufacturer's protocol. The quality of the total RNA was verified using a 2200 TapeStation RNA Screen Tape device (Agilent, Santa Clara, CA, USA) and its concentration ascertained using an ND-1000 spectrophotometer (NanoDrop, Wilmington, DE). Ribosomal RNA was removed using the Ribo-Zero rRNA Removal Kit (Bacteria) from 2 microgram of Total RNA, and the depleted RNA was used as input in the Illumina TruseqRNA stranded kit without PolyA-enrichment. The prepared libraries were evaluated with the High sensitivity D1000 screen Tape (Agilent Tape Station 2200). The indexed libraries were quantified with the ABI9700 qPCR instrument using the KAPA Library Quantification Kit in triplicates, according to the manufacturer's protocol (Kapa Biosystems, Woburn, MA, USA). Five μL of the pooled library at a final concentration of 2 nM were used for sequencing using Illumina Miseq with a 150 Paired end-Read sequencing module.
GEO Accession
GSM3446282