title: RNAseq of Malpighian tubules: freshwater sample 2
design: Total RNA samples were screened with an Agilent Bioanalyzer 2100 to confirm quality and concentration. Each sample was diluted to 250 ng for input into NEBNext Poly(A) mRNA Magnetic Isolation Module (E4790, New England Biolabs), followed by NEBNext Ultra II Directional RNA Library Prep kit for Illumina (E7760). The NEB protocol was followed with the following adjustments: 0.8 bead clean after second strand synthesis, adaptor dilution of 1:30, 0.7 bead clean after ligation, 15 cycles of amplification, and dual bead clean after enrichment. Standard Illumina adaptors were used for all, but each library was barcoded independently with TruSeq indexes. Final libraries were qualified and quantified with Agilent Bioanalyzer 2100, then pooled equimolarly. Sequencing was performed with Illumina NextSeq500 using a High-output Single-end 75 bp kit.