RNA-Seq of Nephila clavipes: adult female dissected silk gland pair (07)
Strand-specific, ribosomal RNA (rRNA)-depleted paired-end RNA sequencing libraries were constructed for RNA extracted from silk gland pairs using NEBnext Ultra Directional RNA Library Prep Kits for Illumina ((New England Biolabs) and TruSeq RNA sequencing adapters (Illumina). To eliminate DNA content, total RNA was treated with TURBO-free DNase (Life Technologies), then purified by ethanol precipitation. Ribosomal RNA content was depleted using the Ribo-Zero Gold Kit (Epicentre, Human/Mouse/Rat), then purified by ethanol precipitation. The DNase-treated, rRNA-depleted RNA was then used as input for the NEBnext Ultra Directional RNA Library Prep Kit, and libraries were constructed using protocol ŇBÓ (specific to rRNA-depleted RNA, 2.0), and followed all default volumes, recipes, and reaction parameters (New England Biolabs). TruSeq RNA adapters (Illumina) were used to uniquely barcode each RNA sequencing libraries, which were subsequently PCR enriched with IlluminaŐs TruSeq PCR Master Mix and Primer Mix and the cycling parameters outlined in the NEBnext protocol. After PCR enrichment, final RNA libraries were cleaned using AMPure XP beads at a 1:1 ratio. Please refer to associated publication for details on dissection methods of specific tissues.