7 protocols
Illumina base calling using PHRED scoring for quality assesment
Each library was loaded in a single lane and sequenced in a Ilumina GAIIx sequencer with a 36 cycles single read sequencing kit.
Two flasks containing 10 mls of fresh Brucella Broth were (each one) inoculated with 2 mls of an overnight culture of Brucella abortus 2308.
Erythritol was added at 1 mg/ml to one of them and both cultures were then allowed to grow during 2.5 h until an OD600 = 0.6-0.7
RNA was purified by using the Qiagen RNeasy Protect bacteria mini kit (Quiagen, USA)and RNase free DNase I digestion. Pure RNA (5 ug) was depleted of rRNA with the Ambion MICROBExpress kit.
High quality reads, 36 bp long, were aligned with the B. abortus 2308 reference genome (PRJNA62937ID) using MAQ allowing up to 2 missmatches. Number of reads were obtained with MAQ-pileup and converted to RPKM. Differential expression was assumed for a foldchange >2 between conditions.
library generation
Libraries were generated using the Illumina mRNA-seq sample preparation kit. Retrotranscribed dsDNA fragments of 200 bp were gel selected and amplified for 15 cycles