REPLI-g WTA Single Cell Kit (96)

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Protocol tips
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Publication protocol

Generation of Metatranscriptomes: We processed two samples for metatranscriptome sequencing; the single sample that tested positive for EVEV (4.1) and a sample that was negative for EVEV (1.1). Following the manufacturer’s protocol, the GeneRead rRNA Depletion Kit (Qiagen, Inc., Hilden, Germany) was used to reduce the burden of C. cedecei vector DNA and RNA. Depleted samples were then processed with the REPLI-g Single Cell Whole Transcriptome Amplification (WTA) kit (Qiagen, Inc.), according to the manufacturer’s protocol. After review of the REPLI-g nanopore data, it was determined that a comparison to another WTA method for nanopore metatranscriptome sequencing was prudent (additional details in Results). Following the manufacturer’s protocol, we also processed the raw TNA sample with the WTA2 Complete Whole Transcriptome Amplification kit from Sigma-Aldrich Inc. (St. Louis, MO, USA). WTA products (from either kit) were purified with Agencourt (Beverly, MA, USA) AMPure® beads as follows; 1.8x the eluted WTA product volume (54 µl) of AMPure beads was added to the WTA-product (30 µl) and pipette-mixed 10 times. The reaction was placed on a magnetic stand for 10 minutes and the cleared solution was aspirated away. The cDNA-bound magnetic beads were washed 2x with 200 µl 70% ethanol and allowed to air dry for 5 minutes. 40 µl of dH2O was added to the washed beads and pipette-mixed 10 times. The sample was placed back on the magnetic stand for 10 minutes. The purified, eluted cDNA was transferred to a fresh 1.5 ml tube and quantified with a Qubit flourometer (ThermoFisher Sci., Waltham, MA, USA) according to the manufacturer’s protocol.

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Manufacturer protocol

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