RNA-seq of Apis mellifera whole bees taken from a Florida swarm trap
One set of samples with environmental virus, 7 ng of total RNA was used done directly library construction using NEBNext Ultra II Directional RNA Library Prep Kit (New England Biolabs, catalog #E7760). Second set of samples, 200ng of total RNA was used first for ribosomal depletion using by Illumina Ribo-Zero Plus rRNA Depletion Kit following the manufacturers protocol and eluted into 5 ul EB buffer. Then RNA-seq library is processed using NEBNext Ultra Directional RNA Library Prep Kit for Illumina (NEB, USA) following manufacturers recommendations. For both sets of samples, first strand synthesis reaction mix (NEBNext First strand synthesis Reaction Buffer (5X) and NEBNext Random primers), fragmented by heating at 94 C for desired time. This step is followed by first strand cDNA synthesis using reverse transcriptase and oligo dT primers.  Synthesis of ds cDNA is performed using the 2nd strand master mix provided in the kit, followed by end-repair and adaptor ligation. Finally, library is enriched (each library has a unique barcode, each primer has a common adaptor sequence which was added in previous adaptor ligation step, and an overhang unique index unique to each sample) by certain number of cycles of amplification, and purified by Agencourt AMPure beads (Beckman Coulter, catalog # A63881). The library size and mass were assessed by analysis in the Agilent TapeStation using a DNA5000 Screen Tape.  Typically, a 250-800 broad library peak was observed with the highest peak at ~500 bp. Finally, the libraries were pooled equimolarly and sequenced on the Illumina NovaSeq X Plus platform using a 10B flow cell with a 2  150 format. The final library loading concentration was 130 pM with 5% PhiX spike-in as a control. FastQ files were generated using the BCL2fastQ function in the Illumina BaseSpace portal. RNASeq libraries were constructed at the UF ICBR Gene Expression Core (https://biotech.ufl.edu/gene-expression-genotyping/, RRID:SCR_019145). Sequencing was performed at the ICBR NextGen Sequencing (https://biotech.ufl.edu/next-gen-dna/, RRID:SCR_019152).
SUB16261433
6591W