small RNA sequencing of CHO-DXB11 rec
Total RNA samples were isolated using Trizol reagent (Invitrogen, Carlsbad CA) according to the manufactuer’s recommendations. Quality of total RNA was controlled using Nanodrop (Thermo Scientific) and Bioanalyzer (Agilent) analyses, where RNA integrity numbers were required to be >9 for subsequent library preparation: therefore, small RNA fragments of 18 to 36 nucleotides were purified from 10 µg of total RNA on a 15% TBE Urea RNA Gel (Invitrogen, Carlsbad, CA). Apart from this intital purification, Illumina sequencing libraries were prepared according to the Illumina v1.5 preparation kit. Quantities of all libraries were analysed by a fluorescence-based method using the Quant-iT PicoGreen dsDNA kit (Invitrogen) and the Tecan Infinite 200 Microplate Reader (Tecan, Germany) according to the manufacturer’s instructions. The average fragment size of each library was measured by a DNA 1000 LabChip using the 2100 Bioanalyzer (Agilent Technologies, Germany). The molar concentration of each library was calculated from the average fragment size and the corresponding quantity. Subsequently, the libraries were diluted to 1 nM stock solutions with elution buffer EB (Qiagen GmbH, Hilden, Germany). Eventually, 120 µl of a 6 pM dilution of each library were applied to cluster generation using the Single-Read Cluster Generation Kit v2 on the Cluster Station (Illumina Inc., San Diego, USA) according to the manual provided by the manufacturer (Part # 1006080 Rev A) applying the Single-Read Multi-Primer One-Step protocol. Thereby, each library was amplified in a separate lane of the flow cell including the PhiX control in lane no. 5. After cluster generation, the flow cell was sequenced on the Genome Analyzer IIx using one SBS Sequencing Kit v3 generating 36 bp single-reads.