GSM2885058: DT3017pDT16r1; Streptomyces coelicolor A3(2); RNA-Seq
RNA isolation was performed using a modified version of the modified Kirby mix protocol as follows: 14 g of glass beads were added to defrosted pellets along with 5 ml of 30 °C modified Kirby mix (in 50 mM TrisHCl ph8.3; 1% w/v sodium-triisopropylnaphthalene sulphonate, 6% w/v sodium 4-aminosalycilate, 6% phenol mixture) before being vigorously vortexed for 2 minutes. Acid phenol: chloroform 1:1 was then used to isolate nucleic acids before being precipitated with 4 M acetate and 100 % isopropanol. Nucleic acids were subject to an ethanol wash before final resuspension in water. Nucleic acids were repeatedly treated with DNase I using conditions described by the vendor (Roche) until DNA could no longer be amplified using polymerase chain reaction (PCR). RNA was then enriched for mRNA using MICROBExpressTM Bacteria beads, as described by the manufacturer (Ambion). mRNAs were fragmented with ultrasound before antarctic phosphatase treatment and re-phosphorylation with polynucleotide kinase. RNA fragments were then poly(A)-tailed using poly(A) polymerase and an RNA adapter was ligated to mRNA 5' phosphate. First strand cDNA synthesis was performed using an oligo(dT)-adapter primer and M-MLV reverse transcriptase before PCR amplification. The cDNA samples were pooled and size fractionated in the range of 250-500 bp and run on an Illumina HiSeq 2000 system with a 100 bp read length.
GEO Accession
GSM2885058