RNA, forward reads
The RTS DNase kit (MO BIO Laboratories, Carlsbad, CA, US) was used to remove any contaminating genomic DNA from the RNA preparations by mixing 17 l of the RNA extract with 2 l of 10X RTS DNase Buffer and 2 l of RTS Dnase. Reverse transcription (RT) of RNA into single stranded cDNA was performed using RT PCR mix containing: 12 ul of DNase treated RNA, 1.0 l random hexamer primer (10 pmol), 1 l dNTP (10 mM), 0.5 l DEPC treated water, 4 l 5X reaction buffer, 0.5 l Ribolock RNase inhibitor, and 1 l RevertAid premium reverse transcriptase (all components from Thermo Fisher Scientific, Waltham, MA, US). The reverse transcriptase incubation conditions were as follows: 25C for 10 minutes, 50C for 30 min, 85C for 5 min. After the RT reaction, all samples were stored at -80 C until further use. Primer pair 515F/806R targeting the V4 region of 16S rRNA gene was used. PCR amplifications were performed with primers containing template-specific sequences extended by a 2-nt linker and 46-nt barcode. Each of three independent 10 L reactions per DNA sample contained 2 L of 5x polymerase buffer, 1 L of 10 mg/mL-1 bovine serum albumin, 0.5 L of each primer (0.01mM), 0.2 L of dNTPs (10mM), 0.1 L of PCRBIO HiFi Polymerase (PCR Biosystems, London, UK), 0.5 L of template DNA (DNA concentration 10100 ng/L), and 5.2 L of H20. The cycling conditions were 95 C for 1 min; 30 cycles of 95 C for 15 s; 50 C for 20 s; and 72 C for 20 s, followed by 72 C for 5 min. PCR products were purified using the HighPrepTM PCR clean up system (MAGBIO, Gaithersburg, MD, USA) and fluorometrically quantified using the Qubit. PCR products were mixed equimolarly and Illumina adapter sequences were ligated on amplicons using TruSeq DNA PCR-Free LT Sample Prep Kit (Illumina, San Diego, CA, USA).