GSM8827448: Dorsal skin, sample 6; Acipenser transmontanus; RNA-Seq
RNA was harvested in RLT Plus lysis buffer (Qiagen) and extracted with the RNeasy Plus Mini kits (Qiagen) using gDNA eliminator columns. RNA was extracted from tissue in the same way after disruption with an Omni Bead Ruptor 24. RNA integrity was verified by Nanodrop 1000 befor library contsruction. cDNA and SMRTbell libraries were constructed using 300 ng of total RNA for cDNA construction with the NEBNext® Single Cell/Low Input cDNA Synthesis & Amplification Module (New England Biolabs, Ipswich, MA; Cat. #E6421L). After 15 cycles of PCR amplification, cDNA was purified using 0.86X SMRTbell cleanup beads. cDNA libraries were pooled for SMRTbell library construction using the SMRTbell prep kit 2.0 or 3.0 (Pacific Biosciences, Menlo Park, CA) with repair and A-tailing, overhang adapter ligation, and nuclease treatment. The Iso-Seq SMRTbell library was sequenced at UC Davis DNA Technologies Core (Davis, CA) using one 8M SMRT cell (Pacific Biosciences, Menlo Park, CA; Cat #101-389-001), Sequel II sequencing chemistry 2.0, and 24-hour movies on a PacBio Sequel II sequencer. cDNA preparation, amplification, and library prep were performed with the Iso-Seq Express Oligo Kit (PacBio) and SMRTbell Prep Kit 3.0 (PacBio).