GSM6523973: Whole cell, grown to mid log phase, RNA replicate 1 [SC6_1]; Streptomyces clavuligerus; RNA-Seq
Samples were centrifuged, supernatant removed and resuspended in 300 µL TE buffer (Tris-HCl 50 mM, EDTA 20 mM) with 100 mg\mL lysozyme and incubated at 30oC for 30 minutes.  To each sample, 50 µL 10% SDS was added and mixed, then 85 µL of 5M NaCl and mixed.  Trizol was added (1 mL) to each, vortexed for 15 seconds and centrifuged for 10 minutes.  The top, clear, colourless aqueous phase was removed and combined with 300 µL chloroform, vortexed and centrifuged.  The upper aqueous phase was removed and passaged through Qiagen RNeasy kit following the manufacturers instructions.  On column DNase treatment (using Qiagen RNase free DNase) following manufacturers instructions (in brief: following first was with RW1, 10 µL DNase was added to 70 µL RDD buffer and incubated on column at RT for 20 minutes). Samples were resuspended in 90 µL RNase free water and an off column DNase Treatment carried out (following manufacturers instructions: to each sample, add 7.5 (in brief: following first was with RW1, 10 µL DNase was added to 70 µL RDD buffer and incubated on column at RT for 20 minutes) RDD buffer and 2.5 µL DNase, incubate at room temperature for 20 minutes, add 100 µL RLT buffer and 100 µL 90% EtOH and apply to fresh column).  Samples were passed through a second round of RNeasy kit extraction minus DNase steps.  Samples were then submitted to Novogene for sequencing and the following broad steps taken. Messenger RNA was purified from total RNA using poly-T oligo-attached magnetic beads. After fragmentation, the first strand cDNA was synthesized using random hexamer primers followed by the second strand cDNA synthesis. The library was then end repaired, poly A-tailed, adapters ligated, size selected, amplified, and purified and sequenced. 