S1000 cycler

A total amount of 1µg of RNA was in-vitro transcribed into cDNA by using 1 µL dNTP-Mix (10 mM), 1 µL Oligo(dT)18 Primer (0.5 µg/µL), 4 µL RT Buffer (5×), 1 µL Maxima H Minus Reverse Transcriptase (200U/µL), and 0.5 µL RiboLock RNase Inhibitor (40U/µL) (all Thermo Fisher Scientific) on a S1000 cycler (BioRad) at 52 °C for 0.5 h. Further, qRT-PCR runs were performed on a 384-well C1000 cycler (BioRad). In general, all samples were analyzed in technical triplicates using 7.34 ng of cDNA for each replicate and the SYBR-green-based Luna Universal qPCR Master Mix (New England Biolabs, Frankfurt am Main, Germany). At the end of each run, melting curve analyses were performed. Oligonucleotide sequences are given in Table 1 (Table 1).

The V3-V4 region of the 16S rRNA genes was PCR amplified from a DNA aliquot of the extracted gut sample using the forward primer 341F (5′-CCTAYGGGRBGCASCAG-3′) and the reverse primer 806R (5′-GGACTACNNGGGTATCTAAT-3′) synthesized at Invitrogen (Invitrogen, Carlsbad, CA, USA). A unique 12 bp barcode to each primer was used to tag the samples. PCR reactions contained 25 μL 2 × Premix Taq (Takara, Dalian, China), 1 μL each primer (10 mM) and 3 μL DNA (20 ng/μL) template in a volume of 50 µL. They were amplified under the following conditions: 5 min at 94 °C for initialization; 30 s denaturation for 30 cycles at 94 °C, 30 s annealing at 52 °C, and 30 s extension at 72 °C; followed by 10 min final elongation at 72 °C in a Bio-Rad S1000 cycler. The length and concentration of the PCR product were detected using 1% agarose gel electrophoresis. The samples with bright main strip between 400–450 bp were used in further experiments. The PCR products were mixed in equidensity ratios according to GeneTools Analysis Software (Version4.03.05.0, SynGene). Thereafter, the mixed PCR products were purified with the EZNA Gel Extraction Kit (Omega, Norwalk, CT, USA).