GSM2441082: 30-RNAseq; Leptospira interrogans serovar Manilae; RNA-Seq
Cells were collected by centrifugation for 15 min at 4,000 x g. Pelleted cells were resuspended in 700 μl of TRIzol, and RNA was purified according to the manufacturer's instructions (Invitrogen, Saint Aubin, Ile de France, France). DNA was degraded by using a Turbo DNA-free kit according to the manufacturer's instructions (Invitrogen). One microgram of DNase-treated RNA per sample was used to generate cDNA with the iScript cDNA synthesis kit according to the manufacturer's instructions (Bio-Rad, Marnes-la-Coquette, France). Ribosomal RNA was depleted by specific rRNA modified capture hybridization approach ("MicrobExpress" kit, AM1905, Ambion), allowing an enrichment of messager RNA. cDNA librairies were prepared by ligation with an excess of 5 adapter (Illumina TruSeq Small RNA kit) and by reverse transcription using a random primer (RPO primer: 5 ′ CCTTGGCACCCGAGAATTCCANNNNNN-3). The first strand cDNA/RNA hybrids were then run on a 2% Low Range Agarose (Biorad). cDNAs ranging from 120 to 250 bp were extracted from a gel slice by using the Qiaquick gel extraction kit (Qiagen) and PCR amplified for 14 cycles using the Illumina primer RP1, and one of the indexed primers (Illumina TruSeq Small RNA kit). The resulting PCR products were purified with Agencourt AMPure Beads XP (Beckman).
GEO Accession
GSM2441082