B22
miRNAseq_homo_sapiens_Sample_10
To prepare samples for small-RNA sequencing, total RNA was extracted using an AllPrep nucleic acid extraction kit (Qiagen, Valencia, CA). RNA was quantified using the RiboGreen fluorometric assay (Thermo Fisher, Waltham, WA). RNA integrity was then measured using a model 2100 Bioanalyzer (Agilent, Santa Clara, CA). Library creation and sequencing were performed by the Mayo Clinic Genome Analysis Core. Briefly, small-RNA libraries were prepared using ug of total RNA per the manufacturers instructions for the NEBNext multiplex small-RNA kit (New England Biolabs, Ipswich, MA). After purification of the amplified cDNA constructs, the concentration and size distribution of the PCR products were determined using an Agilent (Santa Clara, CA) Bioanalyzer DNA 1000 chip and Qubit fluorometry (Invitrogen, Carlsbad, CA). Four of the cDNA constructs are pooled, and the 120- to 160-bp miRNA library fraction is selected using Pippin Prep (Sage Science, Beverly, MA). The concentration and size distribution of the completed libraries were determined using an Agilent Bioanalyzer DNA 1000 chip and Qubit fluorometry. Sequencing was performed across 4 lanes on an Illumina HiSeq 2000 instrument (paired end).
B22
SUB4539299
B22