Small RNA from peripheral blood from 2-day old zebrafish embryos, wild type
Small RNA libraries were prepared from total RNA (0.5 g) isolated from peripheral blood of wild type (WT) and miR-144-/- embryos at 48 hours post-fertilization. Small RNAs were size-selected in 10% denaturing polyacrylamide gel in 1X TBE in range from 18 to 75 bp. RNA extraction from the gel was achieved by incubation in 0.3 M NaCl overnight at 4C with following precipitation with isopropanol. Pre-adenylated 3 custom adaptor (WT: /5PHOS/N*NCACAAGATCGGAAGAGCACACGTCTGAACTCCAGTCAC/3ddC/; miR-144-/-:/5PHOS/N*NAGAGAGATCGGAAGAGCACACGTCTGAACTCCAGTCAC/3ddC/) were ligated to small RNAs by T4 RNA ligase for 2 h at 22 C. After ligated products were size-selected by gel from non-ligated adaptor. Reverse transcription was performed by SuperScript III Reverse Transcription kit (Thermo Fisher) using custom RT Cloning Primer (/5PHOS/NNNNAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTAGATCTCGGTGGTCGC-(SPC18)-GGATCC-(SPC18)-GTGACTGGAGTTCAGACGTGTGCTC). Then cDNA products were isolated by gel selection and then circularized by CircLigase ssDNA Ligase (Epicentre). cDNA libraries were amplified in reactions containing forward and reverse Illumina index primers.