Comparative assessment of SNP and microsatellite markers for assigning parentage in a socially monogamous bird
RNA-seq library was generated by pooling 25 µL of whole blood preserved in RNAlater obtained from 10 individuals sampled during fall migration in 2013 at the Braddock Bay Bird Observatory, NY. 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. The two barcoded cDNA libraries were pooled in equimolar ratio and sequenced on a single lane using 100 bp single-end reads on an Illumina HiSeq 2000 at the Cornell University Core Laboratories Center