Qubit fluorometer

Total RNA was isolated using the High Pure RNA Isolation Kit (Roche Life sciences,Basel, Switzerland) following the manufacturer’s instructions. The integrity was checked with bio-analyzer (Agilent 2100 Technologies, USA) to yield high quality RNA for further processing. The mRNA was extracted and purified from the total RNA by using TruSeq stranded mRNA HT sample preparation kit (Illumina Inc., U.S.A), followed by purity check with Qubit fluorometer before proceeding to cDNA synthesis.

16S rRNA amplicon sequencing was performed targeting the V3-V4 hypervariable region. Negative and positive control samples were included in the sequencing: negative DNA extraction control, negative PCR control, and a mock community (ZymoBiomics microbial community DNA standard, Zymo Research, Irvine, California, USA) as a positive control. Amplicon libraries were generated following the Illumina protocol (https://support.illumina.com/documents/documentation/chemistry_documentation/16s/16s-metagenomic-library-prep-guide-15044223-b.pdf) with the exception that increased amount of 75 ng of DNA template was used in the amplicon PCR reaction. Amplicon libraries were quantified using Qubit fluorometer and 10% of Phix (Illumina, San Diego, California, USA) was added to each equimolar pool. Sequencing was performed using MiSeq Reagent Kit v3 and paired-end 2×300 bp protocol on a MiSeq System (Illumina).

We added to each sample, 2 μL of universal i5 primer, 10 μM of barcoded i7 primers (EpiCypher), and 25 μL of CUTANA High Fidelity 2x PCR Master Mix (EpiCypher). Primer sequences are listed in S4 Table. The following PCR settings were used: 58°C for 5 min, 72°C for 5 min, 98°C for 45 sec, then cycle at 98°C for 15 sec, 60°C for 10 sec, and 72°C for 1 min. Samples were amplified for 18 cycles. Size selection was performed using Ampure XP beads following the manufacturer’s recommendation. Libraries were eluted in 15 μL of elution buffer (Qiagen) and quantified using both a Qubit fluorometer and qPCR against Illumina primers. Libraries were sequenced in 75 bp paired-end sequencing format (Illumina NextSeq). Processing time for this protocol is 1.5 days.

DNA was amplified using the No. 5 Hot Mastermix 2.5x kit (5 PRIME, Hilden, Germany) with bacteria/archaeal primers 515F/806R specific for the hypervariable V4 region of the 16S rRNA gene (Caporaso et al., 2012 (link)). The forward and reverse primers were modified to incorporate a 12 bp Golay error-correcting barcode that enables sample multiplexing (Caporaso et al., 2012 (link)). All samples were amplified in triplets and pooled after PCR amplification (94°C for 3 min; 35 cycles of 94°C for 45 s, 50°C for 1 min, 72°C for 1.5 min; 10 min rest to finish). The PCR product was run on a 1% agarose gel and the DNA concentration was estimated with a Qubit fluorometer (Invitrogen, Carlsbad, CA, United States). The amplicons were pooled at equimolar concentrations and purified with the Ultra Clean PCR Clean-Up kit (MoBio, Carlsbad, CA, United States) following the supplier’s instructions. The DNA concentration of the pooled amplicon product was measured with a Qubit fluorometer and adjusted to 2 nM. The library was denaturated and diluted as described by Illumina (MiSeq System User Guide, Part # 15027617 Rev. C), before it was loaded onto a MiSeq cartridge (Illumina, San Diego, CA, United States) and sequenced using a 500 bp paired-end sequencing protocol.

50–100 ng/sample RNA from each animal was used as input for cDNA synthesis and amplification with the SMARTer Ultra Low Input RNA kit (Clontech, CA) following manufacturer recommendations with 13 cycles of PCR amplification. cDNA quantity and quality were evaluated with a Qubit fluorometer and TapeStation 4200, respectively.
100pg of amplified cDNA was used to generate DNA libraries using Illumina Nextera XT DNA Library Kit (Illumina, CA). Twelve library amplification cycles were applied per manufacturer’s recommendation. Library quantity and quality were evaluated as described above for the cDNA. Illumina paired-end 151 bp sequencing was performed on a Next-seq 500 apparatus (Illumina, USA).