We use a synthetic RNA spike-in as a control in each of our wells. The sequence is meant to mimic the actual SARS CoV-2 amplicon (same amplicon structure except for a 6 nt unique stretch to distinguish). The use of this synthetic spike in has two important advantages.
We RT-PCRed synthetic SARS CoV-2 RNA from Twist, adding on a T7 promoter in order for us to in vitro transcribe the spike RNA. For the primers used in spike construction, look here. Below are .gb files of the T7 spike in templates. NOTE: Please consult the primer contamination page about our experience getting contaminated oligos when ordering the T7 template primer for the N1 spike.
s2_synthetic-spike_t7-template.gb
n1_synthetic-spike_t7-template.gb
For in vitro transcription, we used the NEB HiScribe kit. The protocol we used is below:
After IVT, we DNase treated the reactions, purified the RNA with a Zymo RNA clean and concentrator column, and quantified with an Agilent TapeStation.
We added 100 copies of the spike RNA per reaction into our mastermix before aliquoting. We didn't spend much time optimizing spike concentration, but we wanted to include it at a high enough concentration to ensure accurate quantitation but also not too high to waste sequencing reads.
Before:
https://s3-us-west-2.amazonaws.com/secure.notion-static.com/f8524ac1-f477-407a-b2cd-e5ab62bfdc44/preview(131).pdf
After: