alias: pepp.ochl
title: Ochlerotatus sierrensis from Pepperwood Preserve
design: Individual female mosquitoes representing each species from the different collection sites were selected for SMS sequencing to characterize the microbiota. Prior to processing, whole mosquitoes were washed in 70% ethanol, distilled water and phosphate-buffered saline (PBS) solution to remove external microbes. A similar wash protocol for Drosophila was found to be sufficient to remove external microbes (Chandler et al., 2011). Next, samples were individually homogenized in PBS with steel Lysing Matrix I beads (MP Biomedicals).  Samples were prepared for sequencing by first undergoing total RNA isolation, rRNA subtraction, reverse transcription and random amplification into cDNA libraries. For RNA extraction, we used Masterpure Complete DNA and RNA Purification kit according to the manufacturer’s protocols. After DNase treatment, pellets were resuspended in 30µl of TE buffer and 1µl of RiboGuard was added to each tube. Nanodrop and Bioanalyzer analyses were performed, each using 1µl of sample (data not shown). Following ribosomal subtraction using Ribo-Zero Gold (magnetic beads, human/rat/mouse kit), samples underwent random reverse transcription followed by random amplification. Samples were tagged with a molecular ID tag (MID) and pooled equimolar for a single flow cell of sequencing. Sequencing was done on the Illumina Hiseq 2000 at HudsonAlpha Institute for Biotechnology, using an insert size of 100 basepair paired-end reads.
identifiers.namespace: California Academy of Sciences
identifiers.id: pepp.ochl