title: GSM9662626: Bacillus subtilis subsp. subtilis str. 168 wildtype, biofilm, replicate 1; Bacillus subtilis subsp. subtilis str. 168; RNA-Seq
library_construction_protocol: Biofilms were transferred into a tube and submerged in 800 µL DNA/RNA Shield supplemented with 10 mg/mL lysozyme. To homogenize biofilms were first passed through a 23g1 needle 10-15 times and then sonicated for 12 bursts (duty cycle 20 %, output control 2). 30 min incubation at 37 °C under shaking conditions and subsequent sonification for 4x20 bursts lysed the cells, and debris were pelleted by centrifugation at max. speed for 2 min. To isolate total RNA the protocol of the Quick RNA Miniprep Plus Kit (Zymo Research) was followed. Concentration of isolated RNA was measured with a NanoDrop and integrity was confirmed in a denaturing agarose gel (Aranda et al., 2012). High-quality total RNA aliquots of the biological replicates were rRNA-depleted using Pan-Bacteria riboPOOL (siTOOLs BIOTECH). Successful rRNA depletion was confirmed using Agilent RNA Pico 6000 kit on an Agilent 2100 Bioanalyzer (Agilent Technologies). The TruSeq Stranded mRNA Library Prep Kit from Illumina, starting with fragment, prime, and finish was used to prepare the cDNA libraries. The cDNA libraries were then sequenced in paired-end mode on an Illumina NextSeq2000 system with 50 nt read length and a minimum of 5 million clusters per sample.