small RNA sequencing of Bos taurus
After isolation of total  microRNA using miRNeasy Serum/Plasma Kit (217184, Qiagen), it was quantitated  using the Qubit microRNA kit. The dye binds only to microRNA or other very  small single stranded RNA and is not affected by salts. After Qubit total microRNA  estimation a bead cleanup was conducted. However, there was quite a bit of  sample loss and a modified protocol for microRNA was necessary. For low input  samples, <50ng miRNA test runs are recommended to determine the necessary  loading concentration. For our experiment- 10ng Qubit quantitated microRNA  was used to prepare the library for each sample. Adapter concentrations were  diluted to , 3 ligation went overnight at 20C, and 19 PCR cycles were used  to amplify the library (PCR bias is not a factor when amplifying microRNA  products, as the fragment sizes are the same). The libraries were then  purified by Pippin and QCd using the Qubit and Fragment Analyzer. For  sequencing, each sample was diluted to 2nM, then 10ul of each 2nM sample was  pooled. The total 2nM pool was then diluted to 1.0pM through a series of  denaturation and dilution steps, starting with 10ul of the 2nM pool. We  loaded a total of 1300ul of 1.0pM pool spiked with 10% PhiX for sequencing  instrumentation control on the to NextSeq500. The flow cell was a 150cycle  Mid Output kit and we ran a 75bp Single Read run.