GSM1619937: s_AC30-30min; Homo sapiens; miRNA-Seq
RNA was extracted from tissue using the Qiagen miRNEasy kit following the manufacturer's protocol. Small RNA libraries were prepared according to manufacturer’s instructions for the NEBNext Multiplex Small RNA Kit (New England Biolabs; Ipswich, MA).  Briefly, adaptors were ligated to the 3’ and 5’ ends of the miRNAs as part of total RNA samples.  A complementary primer was annealed to 3’ adaptor sequences followed by reverse transcriptase (Superscript III; Invitrogen; Carlsbad, CA) to generate a cDNA library of small RNAs.  The library was enriched by 12-15 cycles of PCR employing a common 5’ primer and up to 24 unique Index primers (3’ adaptor complement; equivalent to Illumina TruSeq Small RNA sequences).  PCR products were assessed for miRNA products by Agilent Bioanalyzer DNA 1000 (Santa Clara, CA) analysis.  The 130-155bp fraction was quantitated by region analysis to determine equimolar amounts of libraries to pool.  Pooled PCR reactions were purified and concentrated using a Qiagen MinElute protocol and eluted in 30L nuclease-free dH2O. Small RNA libraries were fractionated to extract an miRNA-enriched sample via 3% Pippin Prep gel cassettes (Sage Science, Beverly, MA).  The 125-160bp recovered fraction was purified by Qiagen MinElute and recovered in 10L nuclease-free dH2O. Individual or pooled miRNA fractions were assessed by a second Agilent DNA 1000 assay. A predominant peak at 140-150bp is evidence that the miRNA-modification molecular biology and size selection steps have performed as expected. The final concentration of each library or pool was determined by Qubit fluorometry using the Broad Range or High Sensitivity reagents (Invitrogen). Libraries were then loaded onto paired end flow cells at concentrations of 8-10 pM to generate cluster densities of 700,000/mm2 following Illumina’s standard protocol using the Illumina cBot and cBot Paired end cluster kit version 3.
GEO Accession
GSM1619937