GSM1698369: RNA_S2; Streptomyces coelicolor; RNA-Seq
The mycelium was resuspended in 500 μl lysis buffer composed of 20 mM Tris-HCl (pH 7.4), 140 mM NaCl, 5 mM MgCl2, and 1% Triton X-100. Resuspended cells were dripped into liquid nitrogen and then ground with pestle and mortar. The powdered cells were thawed and centrifuged at 4 °C for 5 min at 3,000 g to remove cell debris. The supernatant was recovered and centrifuged at 16,000 g for 10 min to clarify supernatant. total RNA was isolated using miRNeasy Mini kit (Qiagen) in accordance with manufacturer’s instructions. Genomic DNA was removed by using DNA-freeTM Kit (Ambion), and rRNA was removed by using Ribo-ZeroTM rRNA Removal Kit for Meta-bacteria (Epicentre). 200 ng mRNA was then fragmented by 10× Fragmentation buffer (Ambion). First strand cDNA was synthesized with random primers (Invitrogen), then second strand cDNA was synthesized using dUTP instead of dTTP. The library for Illumina sequencing was constructed using TruSeqTM DNA Sample Prep Kit (Illumina) according to the manufacturer’s instructions with a modification: degradation of the second strand which contains dUTP by treating USER enzyme (NEB) to the library.
GEO
GSM1698369
GEO Accession
GSM1698369