miRNA-Seq of mus musculus: PTH injected bone
500 ng of total RNA was converted into microRNA NGS li braries using NEBNEXT library generation kit (New England Biolabs Inc.) according to the manufacturers instructions. Each individual RNA sample had adaptors ligated to its 3 and 5 ends and converted into cDNA. Then the cDN A wa s pre amplifi ed with specific primers containing sample specific indexes. After 15 cycle pre PCR the libraries were purified on QiaQuick columns and the insert efficiency evaluated by Bioanalyzer 2100 instrument on high sensitivity DNA chip (Agilent In c.). The microRNA cDNA libraries were size fractionated on a LabChip XT (Caliper Inc.) and a band representing adaptors and 15 40 bp insert excised using the manufacturers instructions. Samples were then quantified using qPCR and concentration standard. Based on quality of the inserts and the concentration measurements the libraries were pooled in equimolar concentrations (all concentrations of libraries to be pooled are of the same concentration). The library pool(s) were finally quantified again with qPC R and optimal concentration of the library pool used to generate the clusters on the surface of a flowcell before sequencing using v3 sequencing methodology according to the manufacturer instructions (Illumina Inc.). Samples were sequenced on the Illumina NextSeq 500 system. The system uses quality score binning enabling a more compact storage of raw sequences . Using only 8 levels (Levels: No call, 6, 15, 22, 27, 33, 37,40) of quality the method has been tested and found to virtually loss less. Please ref er to Illumina white paper for further explanation of the quality score binning system.