GSM4787231: BM110poolTAPM; Streptococcus agalactiae; RNA-Seq
15-40 ml of bacterial culture were rapidly centrifuged, the pellet was frozen in dry ice/ethanol and stored at -80 degree centigrade. Bacterial pellets were resuspended in 400 µl 10% glucose, 60 mM EDTA, 12.5 mM Tris pH 7.6. Bacteria cell walls were broken by using two cycles in FastPrep (FastPrep FP120 homogenizer- speed: 6, time:30 s) at 4 \"C, separated by 1 min., in the presence of 0.4 g of 200-300 µM glass beads and 500 µl acid phenol pH 4.5. After centrifugation the aquaeous phase was extracted once with 1 ml trizol and 100µl phenol:chloroform:isoamyl alcohol (25:24:1 volume ratio) and once with 200 μl chloroform:isoamyl alcohol and RNA was precipitated by isopropanol. Traces of DNA were removed by using the Turbo DNA-free kit(Life Technologies) according to manufacturer's instructionq. RNA quality was controled on Agilent 2100 Bioanalyzer with RNA 6000 nanochip and only RNA with RNA Integrity Number (RIN) higher than 9 was used.8 µg total RNA from each RNA sample was depleted in rRNA by using the MICROBExpress bacterial mRNA enrichment kit and rRNA depletion was controled on Agilent Bioanalyzer. ssRNA-seq performed as follows : depleted RNAs from each of the 6 samples were mixed. Depleted RNA samples were divided into two subsamples (hereby TAP+ and TAP-) containing the equivalent of 7.5 μ g of total RNA. The TAP+ subsample was pretreated with 10 U of Tobacco Acid Phosphatase (Tebu-Bio), the other steps being strictly run in parallel for the two subsamples. TAP treatment was followed 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 were purified twice with 1.3 volumes of AMPure beads (Agencourt), DNA concentration was measured using Qubit quantification and size of the PCR products was estimated on DNA 1000 Bioanalyzer chip (Agilent) before sequencing on Illumina GAIIX or HiSeq 2000