Biodrop lite spectrophotometer

RNA was isolated from Control-MO, nrf1a-MO, nrf1b-MO, nrf2a-MO, and nrf2b-MO embryos at 96 hpf in order to examine expression of genes related to glutathione synthesis and induction of the antioxidant response. Briefly, 10 embryos were pooled and collected into RNAlater (Fisher Scientific), and stored at −80 °C until use. A total of 3 samples were collected for each morpholino group, across 3 experimental replicates. RNA was isolated using the GeneJET RNA Purification Kit (Fisher Scientific), following manufacturer instructions. RNA concentrations were quantified using the BioDrop µLITE spectrophotometer (BioDrop), and 500 ng of RNA were reverse transcribed to cDNA using the iScript cDNA Synthesis Kit (Bio-Rad). Sample cDNA was stored at −20 °C until use.

Adult specimens of V. ellipsiformis and U. peninsularis were collected in July 2014 from Straight River (Minnesota, USA; Lat 44.006509, Long −93.290899) and Suwannee River (Florida, USA; Lat 29.58684, Long −82.94095), respectively. Mussels were shipped alive to the Université de Montréal, where they were opened and sexed by microscopic examination of gonad smears. Mature gonads of all unambiguously sexed specimens were frozen at −80 °C until further analyses.
Total RNA was extracted from frozen gonad tissues with RNeasy Plus Universal Mini Kit (QIAGEN Inc., Valencia, CA) and treated with Turbo DNase (AMBION, Austin, TX) following the provided protocols. A total of 25 RNA extractions were performed, 14 for U. peninsularis (8 females and 6 males) and 11 for V. ellipsiformis (7 females and 4 males). The quality and quantity of extracted RNA were examined via electrophoresis on 1% agarose gel and BioDrop µLITE spectrophotometer. Sixteen samples (four males and four females for each species) were sent to the Institute for Research in Immunology and Cancer Institute (IRIC, Université de Montréal, Montréal, Canada) for Illumina library preparation and paired-end (2×100 bp) sequencing (Illumina HiSeq2000; San Diego, CA).