Ocean warming and acidification alter calcification and innate immune system gene expression in juvenile American lobsters, Homarus americanus
RNA isolation was done following the TRI Reagent Protocol and a subsequent DNAse cleanup with a TURBO DNA-free kit (ThermoFisher Scientific, cat# AM1907). Initial RNA quantity and quality was assessed using a ThermoFisher NanoDrop 2000 UV-Vis Spectrophotometer and Agilent 2100 Bioanalyzer. cDNA libraries were constructed using 800 ng of total RNA following Illumina Truseq Stranded mRNA kit and sample preparation guide (Illumina, San Diego, CA). Each sample was labeled with a unique index. Reverse transcription was performed using SuperScript II Reverse Transcriptase (Invitrogen, Carlsbad, CA). Purified cDNA was selected for using AMPure XP beads (Beckman-Coulter, Indianapolis, IN). Library size distribution and quality was examined using an Agilent 2100 Bioanalyzer (CPCT Genomics Core, UMass Boston) and final quantification was performed on a Qubit 2.0 Fluorometer (Life Technologies, Thermofisher Scientific, Carlsbad, CA). Sequencing was performed on an Illumina HiSeq 2500 at the University of Massachusetts Center for Personalized Cancer Therapy (CPCT) Genomics Core to a minimum depth of 30 million reads following the 76 SR protocol with 76 bp single-end reads.