RNA-Seq of Sequencing negative control
Whole viscera were collected from individual fishes by dissection using flame sterilized tools on a laboratory bench sterilized with a 10% bleach solution. Samples were placed into 2ml tubes containing 0.1mm zirconia/silica beads. DNA was extracted from the viscera using the DNeasy Blood and Tissue Kit using methods of the manufacturer instructions (Qiagen, Hilden, Germany) which were modified by adding our reaction to the MP Biomedicals Fastprep 24 machine for 5 minutes at 6.5 M/S to lyse tissue as well as 10ul of lysozyme and 4ul RNase directly after incubation. I also performed quality control measures by using Qubit Fluorometer model Q32857 the Qubit dsDNA HS Assay Kit, following the manufacturer's instructions to quantify DNA yield (Thermofisher Scientific). I used 1.5% agarose gel with ethidium bromide stain to check DNA extraction quality. The DNA extraction steps resulted in a DNA yield of 30ng/L, if this threshold was not met the viscera were re-extracted. If one sample did not yield high enough DNA concentration, it was replaced by another fish specimen from the same site and family. Microbial DNA was sent to the RTSF Genomics Core at Michigan State University for sequencing. The V4 hypervariable region of the 16S rRNA gene was amplified using dual indexed, Illumina compatible primers following the protocol described in Kozich et al. (2013). PCR products were batch-normalized using an Invitrogen SequalPrep DNA normalization plate, and product recovered from the plates were pooled. The pool was concentrated and cleaned up using a QIAquick Spin column and AMPure XP magnetic beads. The pool was quality checked and quantified using a combination of Qubit dsDNA HS, Agilent 4200 TapeStation HS DNA1000 and Invitrogen Collibri Illumina Library Quantification qPCR assays. This pool was loaded onto one MiSeq v2 Standard flow cell, and sequencing was carried out in a 2x250bp paired end format using a MiSeq v2 500 cycle reagent cartridge. Custom sequencing and index primers complementary to the 515f/806r oligomers were added to the appropriate wells of the reagent cartridge. Base calling was done by Illumina Real Time Analysis (RTA) v1.18.54, and output of RTA was demultiplexed and converted to FastQ format with Illumina Bcl2fastq v2.20.0.