title: RNA-seq of EBPR lab-scale reactor sludge Time105 Cycle2
design: Sludge samples (15 mL mixed liquor) were quickly put on ice and centrifuged for 1 minute, at 4C and 4200 x g. The pellet was resuspended in 2 times volume (g pellet:volume) of DNA/RNA shield (R1100-250, Zymo Research) and homogenized by passing 3 times through a needle (26G). The sample was snap-frozen in liquid nitrogen and stored at -80C until RNA extraction. For RNA extraction, samples were thawed on ice, resuspended in 0.5 mL of TRIzol (Invitrogen, Fisher Scientific AG, Switzerland) and incubated for 5 minutes at room temperature. Then, 0.1 mL of chloroform was added, vortexed for 15 seconds and incubated for 2 minutes at room temperature before being centrifuged for 15 minutes at 5500 x g and 4C. The upper phase was recovered and mixed with 400 l of 100% ethanol. The sample was then purified using RNA purification kit (Direct-zol RNA Miniprep, Zymo Research, USA) following the manufacturer recommendations. DNA was removed using TURBO DNA-free kit (Thermo Fisher Scientific, USA) following the manufacturer recommendations. The RNA was purified on magnetic beads (Agencourt RNACleaner XP, Beckman Coulter) following the manufacturer recommendation. Bacterial ribosomal RNA (rRNA) was removed using QiaSeqFastSelect 5S/16S/23S kit (Qiagen) following the manufacturer recommendations on 1 g of total RNA. Libraries were then generated using the Truseq Stranded mRNAribo zero sample preparation kit (Illumina, USA). The sequencing analysis was performed at the Lausanne Genomic Technologies Facility, University of Lausanne (Switzerland) on NovaSeq 6000 in paired-end mode (2x150).