A comprehensive SARS-CoV-2–human protein–protein interactome reveals COVID-19 pathobiology and potential host therapeutic targets
) was cloned into pDEST-AD and pDEST-DB vectors. All AD and DB expression clones were transformed into Y2Hstrains MATa Y8800 and MATα Y8930 , respectively. To screen out autoactivating DB-ORFs, all DB-ORFY8800 transformants and scored for growth on SC-Leu-Trp+3AT and SC-Leu-Trp-Ade plates, where DB-ORFs that triggered reporter activity were removed from further experiments.
-seq, we performed pairwise Y2H testing of each identified pair to ensure high reproducibility.Zymolyase and incubated for 45 min at 37 °C followed by 10 min at 95 °C to prepare yeast cell lysate used as -seq was carried out as previously described. In brief, plasmid from individual wells of 96-well plates were PCR amplified using a plasmid-specific forward primer and a reverse primer consisting of a well-position-specific barcode and TruSeq 3′ sequencing adapter. Amplicons derived from the same 96-well plate were pooled and purified using QIAquick PCR Purification Kit .
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