Gene expression analyses of saliva-derived in vitro biofilms during carbohydrate fermentation and pH stress
Replicate biofilm samples that were collected after 2 hrs of carbon starvation at neutral pH are abbreviated pH 7 Rep. 1, pH 7 Rep. 2 and pH 7 Rep. 3.  Samples collected at 6 hrs and 9 hrs after glucose amendment are abbreviated pH 4.2 Rep. 1, pH 4.2 Rep. 2, pH 4.2 Rep. 3 and pH 5.2 Rep. 1, pH 5.2 Rep. 2, pH 5.2 Rep. 3, respectively. The biofilm/RNAProtect-suspensions for each replicate were immediately mixed by vortexing for 5 s and incubated in room temperature for 5 min prior to centrifugation for 10 min at 5000 x g following manufacturer’s recommendations. The pelleted biofilms were stored at -80°C for one week prior to RNA extraction.  Each replicate (in total 9) was subjected to RNA extraction as follows: pelleted and frozen biofilms were thawed on ice in 350 ul lysis buffer (mirVana RNA extraction kit, Life Technologies, Carlsbad, CA). Pellet-biofilm samples representing two growth wells from the same sampling time point were pooled to generate sufficient amounts of mRNA for sequencing. For each time point 3 replicate samples were prepared. Each replicate was transferred to a bead beating tube (PowerBiofilm RNAisolation kit, MoBio Laboratories Inc., Carlsbad CA) and inserted into a Mini-bead beater (Bio Spec Products, Bartlesville, OK). Samples were homogenized twice at speed setting ‘homogenize’ for 30 s. Samples were put on ice between and after beating events.  Total RNA extraction and purification was performed by using the mirVana RNA extraction kit (Life Technologies) and the RNA Clean and Concentrator™ kit (Zymo Research, Irvine, CA), respectively. DNA was removed from the samples by adding 1 ul (2U) Turbo™ DNase (Life Technologies) and incubation in 37°C for 30 min. After DNA removal, 16S rDNA PCR was performed by using a standard 16S amplification PCR protocol for 32 cycles of amplification to verify that DNA was removed. To remove rRNA in total RNA extracts the RiboZero™Magnetic Kit (Epicenter, Madison, WI) was employed according to manufacturer’s directions. mRNA was purified by using the Zymo RNA Clean and Concentrator™ kit (Zymo Research). RNA concentration and integrity was monitored before and after rRNA removal by using the Agilent RNA 6000 Nano Kit (Agilent Technologies, Inc. Santa Clara, CA) and the Agilent RNA 6000 Pico Kit (Agilent Technologies), respectively.     mRNA library from rRNA-depleted RNA was generated by using random-primed mRNA synthesis methods according to the ScriptSeq™v2 RNA-Seq Library Preparation Protocol (Epicenter). Prior to second strand mRNA synthesis the di-tagged mRNA was purified by using the Agencourt AMPure XP system (BeckmanCoulter). Index-reads supplied with the ScriptSeq Kit were added to the libraries which then were PCR amplified for 15 cycles. RNA-Seq libraries were purified and quantified by using the Agencourt AMPure XP system (BeckmanCoulter) and the Agilent DNA 1000 protocol (Agilent Technologies), respectively. Sequencing of mRNA libraries was performed by using a Illumina HiSeq 2000 platform (100 bp paired end reads).