GSM6806928: atoh1b-MO_V; Danio rerio; RNA-Seq
RNA isolation from sorted cells was performed using the Total RNA purification Micro Kit (Norgen Biotek) following the manufacturer's protocol. mRNA was isolated from 30 ng total RNA by poly-dT enrichment using the NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB) according to the manufacturer's instructions. Samples were then directly subjected to the workflow for strand-specific RNA-Seq library preparation (Ultra II Directional RNA Library Prep, NEB). For ligation custom adaptors were used (Adaptor-Oligo 1: 5'-ACA CTC TTT CCC TAC ACG ACG CTC TTC CGA TCT-3', Adaptor-Oligo 2: 5'-P-GAT CGG AAG AGC ACA CGT CTG AAC TCC AGT CAC-3'). After ligation, adapters were depleted by an XP bead purification (Beckman Coulter) adding the beads solution in a ratio of 1:0.9. Dual unique indexing was done during the following PCR enrichment (14 cycles) using custom amplification primers carrying the same sequence for i7 and i5 index (Primer 1: AAT GAT ACG GCG ACC ACC GAG ATC TAC AC NNNNNNNN ACA TCT TTC CCT ACA CGA CGC TCT TCC GAT CT, Primer 2: CAA GCA GAA GAC GGC ATA CGA GAT NNNNNNNN GTG ACT GGA GTT CAG ACG TGT GCT CTT CCG ATC T). After two more XP bead purifications (1:0.9), libraries were quantified using the Fragment Analyzer (Agilent). For Illumina flowcell production, samples were equimolarly pooled and sequenced 75bp single read on an Illumina HiSeq 2500.