title: GSM2123145: Fourth replicate of four species biofilm; Paenibacillus amylolyticus; Microbacterium oxydans; Stenotrophomonas rhizophila; Xanthomonas retroflexus; RNA-Seq
library_construction_protocol: For RNA extraction, planktonic cells from biofilm-containing wells were gently removed, wells were rinsed three times using phosphate buffer saline (PBS) and then the biofilms were scraped with sterile pipette tips into 1ml of PBS. The biofilm suspensions were immediately centrifuged at 5000g for 5 min and resuspended in 100μl of PBS followed by addition of 500μl Ambion RNAlater (Thermo Fisher Scientific, Waltham, MA, USA). The RNAlater-preserved samples were kept at 4°C ON, after which RNAlater was removed by adding 600μl cold PBS and centrifuged at 8000g for 5 min at 4°C. The pellet was resuspended in 200μl lysozyme solution (20μg/ml) and incubated at room temperature for 10 min with vortexing for 10s every 2 min. After adding 700μl RLT buffer and vortexing for 10s, the obtained solution was transferred into bead-beating tubes (provided by the FastDNA SPIN Kit for soil) and bead beaten using the Savant FastPrep FP120 (Qbiogene) for 30s at setting 5.0, followed by centrifugation at 13000g for 8 min at 4°C. 850μl of supernatant were transferred to a new microcentrifuge tube. 470μl of 100% ethanol was added and mixed by pipetting. 700μl lysate was transferred to an RNeasy mini spin column. Total RNA was then further purified from each biofilm sample using the RNeasy mini kit (Qiagen, Hilden, Germany) according to manufacturer's instructions. DNase treatment to remove genomic DNA was conducted according to the instructions in Ambion DNAfree Kit (Thermo Fisher Scientific), except the incubation time was extended to 1h. The complete removal of DNA was confirmed by RT-qPCR using 20µl SYBR Green reactions on Mx 3000 (Stratagene, Cedar Creek, Texas). The qPCR targeted the 16S rRNA gene with the eubacterial primers EUB338 and EUB518. All qPCR reactions were run in technical duplicates and contained 10µl Brilliant III SYBR Green QPCR Master Mix (Stratagene, Cedar Creek, Texas), 1µl forward primer (final concentration 385 nM), 1µl reverse primer (final concentration 385 nM), 1µl template DNA (100x diluted to avoid PCR inhibitors. The program combined the annealing and extension step: 95°C for 3 min followed by 40 cycles of 95°C for 10s, 60°C for 20s, and a final dissociation curve. The standard curve for bacteria was made from a pure culture of Pseudomonas putida kt2440 (Park and Crowley, 2005; VanGuilder et al., 2008). mRNA was subsequently isolated using the Ambion MicroExpress Bacterial mRNA Purification Kit (Thermo Fisher) following the manufacturer's instructions. The RNA transcripts were prepared using ScriptSeq™ v2 RNA-Seq Library Preparation Kit (Epicentre Biotechnologies, Madison, WI, USA) 50bp single reads on an Illumina HiSeq 2000 (Illumina)
attributes.tag: GEO Accession
attributes.value: GSM2123145