Mini incubator

Protean IEF cell, IPG strips pH 3–10 (7 cm), IEF focusing tray with lid, SDS-PAGE (SDS-Polyacrylamide gel) electrophoresis cell (model Mini-PROTEAN 3) were from Bio-Rad, following the manufacturer’s instructions. The image of the gel after staining was acquired in a PROPIC II DigiLab Genomic Solutions USA. Protein digestion was done in safe-lock tubes of 1.5 mL from Eppendorf (Hamburg, Germany). A vacuum concentrator centrifuge model UNIVAPO 150 ECH SpeedVac and a vacuum pump model UNIJET II were used for sample drying and sample pre-concentration. A mini incubator from Labnet was used for gel washing, for protein reduction and for protein alkylation steps. The centrifuge MPW-350 and MPW-65R were from MPW Med. Instruments. Absorption spectra of samples were recorded as microliter samples using a NANODROP ND-1000 Spectrophotometer from Thermo Scientific (USA). Protein identification was done in an Ultraflex II MALDI-TOF/TOF instrument from Bruker Daltonics.

Mixtures of blocking agent, blocking oligos, and indexed koala libraries were heated to 95°C to separate the DNA strands [14] (link). One aliquot from each index library was mixed with streptavidin beads bound with biotinylated KoRV PCR products. Samples were incubated for 48 hours at 65°C under rotation in a Labnet mini incubator. After 48 hours the beads were washed and the hybridized libraries eluted by heating. The DNA concentration was measured by quantitative PCR (qPCR), and the eluted libraries were further amplified accordingly using P5 and P7 Illumina outer primers. The products were then pooled at equimolar concentrations for paired-end sequencing on an Illumina MiSeq platform at the National High-Throughput DNA Sequencing Center, University of Copenhagen.

Humidified chamber comprising a Labnet Mini incubator (Labnet International, I5110A), containing a glass bowl with 25 ml water.