title: RNA-seq of Glaucomys volans: adult male brain
design: For RNA-seq, we extracted and submitted 404-655 ng of total RNA input with high RNA integrity number (RIN) scores (criterion RIN > 8, range 9.1-9.8) from each sample. The quantity of input RNA was measured using a Bioanalyzer 2100 RNA Nano chip (Agilent Technologies, Santa Clara, CA, USA), and the concentration was measured with a Qubit RNA HS Assay on a Qubit fluorometer (Thermo Fisher). Library preparation was performed following the New England Biolabs NEBNext Ultra Directional RNA Library Preparation protocol. Between 404-655 ng of RNA input was enriched for poly-A mRNA and incubated for 4 min at 94C to fragment into a 200-300-base range. RNA fragments were converted to double-stranded cDNA and end-repaired and adenylated at 3 with overhang A, to ligate with overhang T Illumina adapters. The cDNA library fragments were amplified under the following PCR reaction conditions: (1) denaturation at 98C for 10 sec, (2) 10 x 98C for 10 sec, (3) 60C for 30 sec, (4) 72C for 30 sec, and (5) an extension step of 72C for 5 min. During the amplification step, each of the samples were amplified with different barcode adapters for multiplex sequencing. After amplification, cDNA library size was measured by a Bioanalyzer 2100 DNA High Sensitivity chip (Agilent Technologies) using 1 l of the final cDNA library. The cDNA libraries were quantified by qPCR using the Kapa Library Quantification Illumina/ABI Prism Kit protocol (KAPA Biosystems, Massachusetts, USA).