GSM4944133: RNAseq_E3_D1_GLB426; Bacillus subtilis; RNA-Seq
10 mL of culture were collected and mixed with 1 mL of 10:1 ethanol/phenol stop solution by rapid inversion. Cells were pelleted at 3000 rcf for 10 min at 4C. The supernatant was decanted and cell pellets stored at -80C until RNA extraction. RNA was extracted using the RNeasy plus kit with gDNA eliminator column (Qiagen) following manufacturer's instructions. Ribosomal RNA was depleted using the MICROBExpress kit (Thermo Fisher). The resulting RNA was fragmented for 1 min 45 s at 95C using RNA fragmentation reagents (Thermo Fisher, AM 8740), dephosphorylated at the 3' end and poly-A tailed using T4 PNK (NEB). Revese transcription was performed using indexed poly-dT primer using SSIII (Thermo Fisher). After reverse transcription, cDNA in the range 100 to 120 nt were size selected, and ligated to an adapter with T4 DNA ligase (NEB). The ligated cDNA was size selected (135 to 155 nt) and PCR amplified.
GEO Accession
GSM4944133