title: GSM3031696: 1371_04con; Homo sapiens; miRNA-Seq
library_construction_protocol: libraries were constructed using Ion Ampliseq Transcriptome Human Gene Expression Kit (Cat#A26325; Thermo Fisher Scientific) per the manufacturer's protocol, at the PrimBio Research Institute LLC (Exton, PA, USA). Briefly, small RNA was enriched with the Total RNA-seq Kit v2 using manufactured recommended protocol.  5 ng small RNA was hybridized with Ion Adapters in a thermocycler for 10 minutes at 65⁰C and 5 minutes at 16⁰C.  Hybridized Small RNA was then incubated overnight at 16⁰C with ligation enzyme mix to ligate the Ion adapters.  The hybridized samples were then mixed with a reverse transcriptase master mix and incubated at 70⁰C for 10 minutes, snap cooled, and incubated 42⁰C for 30 minutes to generate cDNA libraries.  The purified cDNA libraries were then amplified by PCR using Platinum PCR Super-Mix High Fidelity and Ion Xpress RNA Barcode reverse and forward primers with the conditions as follows: Step 1: 94⁰C for 2 mins; Step 2: 94⁰C for 30 sec, 50⁰C for 30 sec, 68⁰C for 30 sec for 2 cycles; Step 3: 94⁰C for 30 sec, 62⁰C for 30 sec, 68⁰C for 30 sec for 14 cycles; Step 4: 68⁰C for 5 mins.