title: GSM1909499: Mock; Homo sapiens; miRNA-Seq
library_construction_protocol: Total RNA was extracted from cells using Trizol reagent. 10µg of total RNA was separated on a 12% denaturing Tris-Urea gel. The ~19-24nt size fraction was excised, purified, and ethanol precipitated. The 3’ linker was ligated using T4RNA ligase2, truncated (NEB). RNA was again fractioned and purified. The 5’ linker was then ligated using T4 RNA ligase (Ambion) followed by another round of size fractionation, purification, and precipitation. Both 3’ and 5’ linkers have randomized ends with four random nucleotides (N) at the insertion site to reduce ligase bias (Jayaprakash et al., 2011). RNA containing both the 3’ and 5’ linkers was then reverse transcribed using SuperScript III (Invitrogen). PCR amplification of cDNA was performed using OneTaq 2x PCR Master Mix (NEB) and barcoded reverse primer for multiplexed sequencing.