GSM1822056
GSM1822056: NEG2VD (miRNA); Homo sapiens; miRNA-Seq
RNA was prepared using RNA/protein spin columns (Clontech).  Lysis solution was added to cells which were lysed by passage successively through 21 and 27 gauge needles.  Individual lysates were applied to RNA spin columns, and RNAs were eluted into nuclease-free water.  Before library construction, RNA quality was assessed. We synthesized miRNA libraries from 1,25(OH)2D3 or vehicle treated cells using total RNA samples and a NEBNext® Multiplex Small RNA Kit (New England BioLabs).  Adaptors were ligated to the 3’ ends of the small non-coding RNAs present in 500 ng of total RNA.  A complementary primer was annealed to the 3’ adaptor sequences followed by ligation of a 5’ RNA adaptor.  A cDNA library was created by reverse transcriptase (Superscript III; Invitrogen) of the adaptor-ligated and annealed small RNA population.  The library was enriched by 15 cycles of PCR employing a common 5’ primer and a 3’ primer containing one of eight index primers (3’ adaptor complement; Index sequences equivalent to Illumina TruSeq Small RNA sequences).  The PCR products were subsequently purified.  The libraries were assessed for miRNA products by Agilent Bioanalyzer DNA 1000 (Santa Clara, CA) analysis.  The 130-160 bp region was quantitated to determine equimolar amounts of vehicle and 1,25(OH)2D3-treated sample libraries to pool.  The pooled small RNA libraries were fractionated to extract a miRNA-enriched sample via 3% Pippin Prep (Sage Science) gel cassettes.  The recovered fraction was purified and reconstituted in 10 mcL nuclease-free dH2O.  Pooled miRNA fractions were assessed by a second Agilent DNA 1000 assay.  A predominant peak at 140-150bp indicated that the miRNA-modification and size selection steps were performed as expected.  The final concentration of each library pool was determined. Libraries were loaded onto single 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 cluster kit version 3.  The flow cells were sequenced as two reads: read 1- 51 cycles using the Small RNA Sequencing Primer and an Index read to demultiplex the samples.  Libraries were sequenced on an Illumina HiSeq 2000 using TruSeq SBS sequencing kit version 3 and SCS version 1.4.8 data collection software.  Base-calling was performed using Illumina’s RTA version 1.12.4.2.
GEO
GSM1822056
GEO Accession
GSM1822056