GSM7648531: miRNA_keloid3; Homo sapiens; miRNA-Seq
a. Add 5 μl total RNA (1 μg) and 1 μl RNA 3'Adapter to a new nuclease-free PCR tube on ice. Gently pipette the entire volume up and down 6 times to mix thoroughly. b. Incubate the tube at 70°C for 2 mins and then immediately place the tube on ice. c. Prepare the following mix in a new, nuclease-free PCR tube on ice. (Ligation Buffer 2, RNase Inhibitor 1, T4 RNA Ligase 2, Deletion Mutant 1, Total Volume 4).  d.Add 1 μl Stop Solution (STP) and gently pipette the entire volume up and down to mix thoroughly. Continue to incubate the reaction tube on the thermal cycler at 28°C for 15 mins and then immediately place the tube on ice. e. Add 1 μl of RNA 5' Adapter to a new, nuclease-free 0.2 ml PCR tube. Incubate the tube at 70°C for 2 mins and then immediately place the tube on ice. f. Prepare the following mix in a tube on ice. g. Add 3 μl of the mix to the reaction from step d. Gently pipette the entire volume up and down to mix thoroughly. Incubate the tube at 28°C for 1 hour and then immediately place the tube on ice. Load 1 μl sample on an Agilent Technologies 2100 Bioanalyze.