Comparative assessment of SNP and microsatellite markers for assigning parentage in a socially monogamous bird
RNA-seq library was generated by combining 15 mg each of liver, muscle, and brain (45 mg total tissue) from one adult male black-throated blue warbler that we collected and preserved in RNAlater (Qiagen) during the 2013 breeding season in the Adirondack Mountains, NY. We used a TissueRuptor (Qiagen) to homogenize the individual tissue pool. We followed the ‘standard’ mRNA extraction protocol as detailed in the Dynabeads® mRNA DIRECT™ kit (Invitrogen). We performed the mRNA extraction protocol twice to maximize the removal of rRNA from our extraction before constructing cDNA libraries. We converted mRNA into cDNA libraries using the NEBNext RNA First Strand Synthesis Module (New England BioLabs). We then performed second strand cDNA synthesis, end repair, dA-tailing, and adaptor ligation for each cDNA library. Following adapter ligation and library purification, we performed 12 cycles of the ‘denaturation annealing extension’ step during the index PCR. We assessed the quality and quantity of cDNA using the QFC algorithm and an Agilent Bioanalyzer 2100.