GSM1630506: 96hpf; Danio rerio; miRNA-Seq
100 μg of total RNA from each sample was isolated using TRIzol® and small RNAs were enriched by differential precipitation using polyethylene glycol. Total RNAs were fractioned using 12% denaturing PAGE and small RNAs of 15-30 nt were gel-isolated using Gel Filtration cartridges from Edge Biosystems. For cDNA synthesis, the small RNA molecules previously isolated were first ligated to a 3' adapter (AMP-5'p-5'p/CTGTAGGCACCATCAATdi-deoxyC- 3') in absence of ATP and gel excised in the range of 35 and 50 nt. A second ligation was performed with the 5' adapter ("Nelson's linker" 5'ATCGTrArGrGrCrArCrCrUrGrArArA 3'), for 1 hour at 37°C, followed by phenol extraction. First strand cDNA synthesis was then performed using a specific 3'-primer and Superscript™ III reverse transcriptase (Invitrogen). RNase H treated cDNA was PCR-amplified with adapter-specific primers. Each sample contained a specific TAG constituted by 3 nucleotides, as detailed next. 24hpf - ATC; 72hpf - ACT; 96hpf - CAG; 5dpf - ATG; 45dpf - CCG; entire adult - GTA; brain - CGG; Heart - CTG; Eyes - GCT; fins - GTT; skin - TAC; gills - TCC. PCR products were then run on 10% denaturing PAGE containing 7 M urea and the corresponding band (100 nt) was eluted from the gel with Probe Elution Buffer from Ambion, at 37°C overnight. These products were used for the emulsion PCR. Parallel DNA pyrosequencing was performed using the Genome Sequencer FLX (Roche), following established protocols for DNA library sequencing.
GEO Accession
GSM1630506