GSM1171547: 16M; Brucella melitensis; RNA-Seq
The cell culture was then collected and RNAprotect Bacteria Reagent (Qiagen, Hilden, Germany) was added to the cultures according to the manufacturer’s instructions. The Brucella RNAs for solexa/illumina sequence were isolated and purified with RNeasy Mini System (Qiagen, Hilden, Germany) according to the manufacture’s protocol. RNA was eluted from the column using RNase-free water. Total RNA was incubated with DNase (Ambion, Foster City, CA) to remove remaining DNA. Subsequently, RNA was purified using two phenol-chloroform extractions and one chloroform extraction, followed by the precipitation of RNA and resuspended in RNAase free TE buffer (10Mm Tris, 1Mm EDTA; pH 8.0; Ambion, Foster City, CA). Each mRNA sample was fragmented into short sequences with divalent cations under elevated temperature. Using these short fragments as templates, first-strand cDNA was synthesized with random hexamer primers and reverse transcriptase (Invitrogen, Carlsbad, CA). The second-strand DNA was synthesized using RNase H (Invitrogen, Carlsbad, CA) and DNA polymerase I (New England Biolabs, Beverly, MA, USA), respectively. The amplified fragments were purified with QiaQuick PCR Purification kit (Qiagen, Hilden, Germany) and resolved with EB buffer for the end preparation and poly (A) addition. The DNA libraries were then prepared according to the manufactuer Illumina’s protocols.