GSM2617025: Bsu_ex_3; Bacillus subtilis subsp. subtilis str. 168; RNA-Seq
Immediately after harvesting cells were treated with TriZOL to isolated total RNA as recommended by the manufacturer. Cell disruption was fascilitated by using FastPrep24 (6 m/s, 30 sec). To precipiated larger RNA species, samples were subjected to high salt precipitation using 5 M LiCl and the supernatant was desalted via G-25 spin filtration (PD-10; GE). RNA was ethanol precipitated and subjected to RiboZero treatment (Samples 3, 6, 9, 12, 15, 18, 23) or processed without further treatment. A 3'-blocked oligonucleotide was adenylated (NEB #E2610) and ligated to the isolated RNA with a truncated T4-RNA-Ligase 2 (NEB #M0242). Ligation products were reverse transcribed (RevertAid; Thermo Scientific) using a 32P 5'-labeled primer and resulting cDNA was isolated from 10% denaturing gel. A pre-adenylated 3'-blocked oligonucleotide was ligated to isolated cDNA using a 5’-App-DNA/RNA-Ligase (NEB #M0319) at conditions given for ssDNA ligation. Ligation products were amplified by PCR in a 100 µL reaction for 15-16 cycles and isolated from MethylBlue stained gel. For each sample a indexed was introduced during PCR and one lane on MiSeq was used per replicate. The third sample from B. subtilis from stationary phase yielded a not sufficient DNA amount and did not generate usable sequence results.
GEO Accession
GSM2617025