title: C.virginica RNAseq library
design: 500ng of total RNA starting material was used for Illumina sequencing libraries for consistent concentrations of starting RNA across all samples. Messenger RNA (mRNA) was isolated from total RNA using NEBNext Poly(A) mRNA Magnetic Isolation kit (New England Biolabs, Inc., Ipswitch, MA, USA) and sequencing libraries were prepared using the NEBNext Ultra RNA Library Prep Kit for Illumina (New England Biolabs, Inc.). Fragment size ranges and library concentrations were determined using the Agilent 2100 Bioanalyzer high-sensitivity DNA chip (Agilent Technologies, Santa Clara, CA, USA). Final library concentrations were quantified using the NEBNext Library Quant Kit Quick Protocol (New England Biolabs, Inc.) and the ViiA-7 Real-Time PCR System (Thermo Fisher Scientific). The KAPA Library Quantification Kit Data Analysis Template (Kapa Biosystems, Inc., Wilmington, MA, USA) was used to calculate Illumina sequencing pooling volumes.