title: GSM8951520: Mixed library SARS-CoV spike pseudotyped virus selected replicate 1; synthetic construct; OTHER
library_construction_protocol: Genomic DNA was isolated from each sample using the JetFlex™ Genomic DNA purification kit (Thermo Fisher, A30700). ACE2 amplicon generation and high throughput sequencing A section of DNA spanning the recombination junction, and including the Kozak sequence preceding the ACE2 open reading frame, was amplified from the genomic DNA extracts using PCR. For each 50 µL PCR reaction, the following components were used: 2 µg genomic DNA (quantified by nanodrop), 0.25 µM  forward primer containing i7 index barcode and 0.25 µM  reverse primer containing i5 index barcode, and 25 µL of Phusion plus high fidelity PCR mix (Thermo Fisher, F548L). A total of five PCR reactions were performed per condition, which each tube initially incubated at 95 °C for 3 minutes, followed by 25 cycles of 95 °C for 15 seconds, 60 °C for 15 seconds, 72 °C for 30 seconds, followed by a final 72 °C incubation for 1 minute. PCR products were mixed with 6x DNA loading dye (NEB, B7024S) and separated on 1.5% agarose TAE gel. Bands corresponding to the expected 293 bp amplicon size were excised and extracted using the Freeze and Squeeze kit according to the manufacturer's instructions (Bio-Rad, 7326166). Eluted PCR products were purified using a Zymo clean and concentrator kit (Zymo Research, D4003). Amplicon concentrations were measured using Qubit dsDNA BR assay kit (Life technologies, Q32853). Amplicons from all the experimental conditions were pooled in equimolar ratios and sequenced on an Illumina NextSeq550 with a 75-cycle high output kit. Oligonucleotide sequences for PCR and custom Illumina primers for sequencing are listed in Supplementary Table 3.