title: Illumina HiSeq 4000 paired end sequencing; Small RNA sequencing of healthy and osteoarthritis diseased anterior cruciate ligament
design: Small RNA sequencing of healthy and osteoarthritis diseased anterior cruciate ligament
library_construction_protocol: Samples were collected in RNA later and stored at -80C  Qiagen miRscript RNA isolation. RNA samples were quantified and the integrity of the RNA was assessed. All samples were used for small RNA-sequencing  on the Illumina HiSeq 4000 platform The nucleic acid library construction utilized the NEBNext® Small RNA Library Prep Set for Illumina® (Multiplex Compatible) but with the addition of a  Cap-Clip™ Acid Pyrophosphatase (Cell script) step to remove any 5' cap structures; i.e.supplied sample was  ligated to 3'adapter as described in the manufacturer's guidelines. After incubation for one hour at 25°C the samples were cleaned with Ampure RNA clean beads with added iso propanol to select for small RNA. Samples were washed with 85% ethanol and resuspended in 25ul water. Samples were incubated with cap clip enzyme in the appropriate buffer for 1 hour at 37°C. The samples were pooled on an equimolar basis and size selected on a Sage Pippin prep using a 3% cassette marker F selecting a range 120bp-400bp. The quantity and quality of the final pool was assessed by Qubit and Bioanalyzer and subsequently by qPCR using the Illumina Library Quantification Kit from Kapa (KK4854) on a Roche Light Cycler LC480II according to manufacturer's instructions.