GSM5732234: Qp_eye tumor 3; Danio rerio; RNA-Seq
Trizol extraction (Invitrogen) 3' Digital gene expression libraries were created by cleaning  RNA samples were cleaned using SPRI beads (1.8X), quantified and quality assessed using an Advanced Analytical Fragment Analyzer. 10ng of total RNA was used for library preparation. 3'DGE-custom primers 3V6NEXT-bmc#1-24 were added to a final concentration of 1 uM. (5'-/5Biosg/ACACTCTTTCCCTACACGACGCTCTTCCGATCT[BC6]N10T30VN-3'  where  5Biosg = 5'  biotin, [BC6] = 6bp barcode specific to each sample/well, N10 = Unique Molecular Identifiers, Integrated DNA technologies), to generate two subpools of 24 samples each. After addition of the oligonucleotides, Maxima H Minus RT was added per manufacturer's recommendations with Template-Switching oligo 5V6NEXT (10uM, [5V6NEXT : 5'-iCiGiCACACTCTTTCCCTACACGACGCrGrGrG-3'  where  iC: iso-dC, iG: iso-dG, rG: RNA G ])  followed by incubation at 42C for 90' and inactivation at 80C for 10'. Following the template switching reaction, cDNA from 24 wells containing unique well identifiers were pooled together and cleaned using RNA Ampure beads at 1.0X. cDNA was eluted with 17 ul of water followed by digestion with Exonuclease I at 37C for 30 minutes, and inactivated at 80C for 20 minutes. Second strand synthesis and PCR amplification was done by adding the Advantage 2 Polymerase Mix (Clontech) and the SINGV6 primer (10 pmol, Integrated DNA Technologies 5'-/5Biosg/ACACTCTTTCCCTACACGACGC-3') directly to the exonuclease reaction. 8 cycles of PCR were performed followed by clean up using regular SPRI beads at 0.6X, and eluted with 20ul of EB. Successful amplification of cDNA was confirmed using the Fragment Analyzer. Illumina libraries were then produced using standard Nextera tagmentation substituting P5NEXTPT5-bmc primer (25μM, Integrated DNA Technologies, (5'-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCG*A*T*C*T*-3'  where  * = phosphorothioate bonds. ) in place of the normal N500 primer.  Final libraries were cleaned using SPRI beads at 0.7X and quantified using the Fragment Analyzer and qPCR
GEO Accession
GSM5732234