GSM2577307: Juvenile phase buds rep1; Malus domestica; miRNA-Seq
Tissues were ground to fine powder, pre-warmed (at 65°C) RNA extraction buffer (2% CTAB, 2% PVP40, 100 mM Tris-HCl, 25 mM EDTA, 2 M NaCl, 0.5 g/L spermidine, 2% β-mercaptoethanol, pH 8.0) was added, and the mixture was incubated for 20 min at 65°C. RNA was extracted by treating the slurry twice with an equal volume of chloroform/isoamyl alcohol (24∶1), and then precipitated with LiCl at a final concentration of 2.5 M. The pellet was dissolved in STE buffer (1 M NaCl, 10 mM Tris-HCl, 1 mM EDTA, pH 8.0) and extracted one more time with chloroform/isoamyl alcohol. RNA was further precipitated with ethanol and dissolved in DEPC H2O. sRNAs of 19-24 nt were gel-purified and ligated to a pair of adapters at the 5’ and 3’ ends by using T4 RNA ligase (NEB). sRNAs with adapters were transcribed into cDNA and amplified by Illumina sequencing-compatible primers.
GEO Accession
GSM2577307