GSM5233525: 154-rep2; Bacillus subtilis PY79; RNA-Seq
10 ml of B. subtilis at OD 0.1-0.2 were centrifuged in 15 ml falcon tubes for 10 min at 4000 rpm. Cell pellets were resuspended in 1 ml of Qiagen RNAprotect Bacteria Reagent, and flash frozen in liquid nitrogen.     Defrosted cells were pelleted by a quick centrifugation. After removing the supernatant, cells were resuspended in 1 ml buffer RLT from Qiagen RNeasy kit. Resuspended cells were transferred in a Fastprep Lysing Matrix B tube (MP Bio) and processed in Fastprep apparatus 45 seconds at speed 6.5M/s. 700 µl of the supernatant, containing lysed cells, was transferred to a new microcentrifuge tube, to which 500 µl of absolute ethanol was added. After vortexing, the lysate was transferred to a RNeasy spin column and centrifuged 15 seconds at > 10000 rpm. RNA purification was then carried out following the instructions of the Quiagen RNeasy kit. RNA quality and integrity were assessed on the Agilent 2200 TapeStation, and RNA concentration was assessed using Qubit RNA HS assay kit. Library preparation was performed using ScriptSeq™ Complete Kit (Illumina, BB1224), for 2 µg of high integrity total RNA (RIN>8). The libraries were sequenced on a NextSeq500 using paired-end sequencing of 75 bp in length.