RNA-seq of Sialia mexicana: embryonic brain
Right hemisphere brain tissue was homogenized by bead-beating at 30Hz with a TissueLyser II instrument (Qiagen) in Qiazol reagent (Qiagen) under chilled conditions, using 0.5mm zirconium oxide beads and two additional 1.0mm beads. RNA was extracted with a miRNAeasy Micro kit (Qiagen 217084) according to the manufacturers instructions. This kit allows for the separate elution of total mRNA and miRNA, which was used in another project. Quality control of total RNA was performed on the Qubit (ThermoFisher Scientific) using RNA broad range reagents (Invitrogen Q10201), and on the Bioanalyzer with the RNA 6000 Nano kit (Agilent 5067-1511; Table SXX). The mRNA library prep was performed using the NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB E7490), the NEBNext UltraExpress RNA Library Prep Kit (NEB E3330), and the NEBNext Multiplex Unique Dual Index Primers (NEB E6446), using 300ng of total RNA input. Lastly, equimolar amounts of each library were pooled and sequenced on the AVITI platform (Element Biosciences) using a Cloudbreak 2x75 high-output kit and flow cell (ElemBio 860-00004), configured for PE 2x75 reads.