RNA-Seq of Apis mellifera: head of young adult treated with Imidacloprid at a target dose of 1000 pg per bee for 48H
Prior to sequencing library preparation, RNA samples  undergo fragment analysis using an Advanced Analytical Technologies Fragment Analyzer and PROSize 3.0 software (v. 4.0.2.7) (Agilent Technologies, Inc., Santa Clara, CA) for quality and quantity measurements.  Each gene expression sample is spiked with the Ambion ERCC Mix 1 (ThermoFisher Scientific, Inc.) at a ratio of 2 L of a 1:1000 dilution to 1 g total RNA, according to manufacturer instructions.   Sequencing libraries from total RNA are prepared using the Illumina Stranded mRNA Kit for mRNA Expression (cat. No 20040534 with IDT for Illumina UDI Indexes set A and B, Cat. No 20040553 and 20040554 respectively).  Briefly, mRNAs are isolated via attachment to oligo(dT) beads, chemically fragmented to a mean size of 150 nt, and reverse transcribed into cDNA via random hexamer priming.  Resulting double-stranded cDNA fragments are end-repaired to create blunt-end fragments, 3 adenine-tailed, ligated with indexed adapters, and PCR-amplified using Illumina primers.  Purified PCR-amplified libraries are checked for quality and quantity on an Agilent TapeStation 4200 using D1000 Screentape before normalization and sample pooling.  Pool is sequenced with an appropriate sized flowcell to target 20-25M reads per sample on either a NextSeq or a Novaseq depending on total sample number and total number of reads needed