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.