title: GSM4827393: SARS-CoV-2 S-RBD lib sorted for increased binding specificity towards the engineered sACE2.v2.4 (Independent Rep 1); Severe acute respiratory syndrome coronavirus 2; OTHER
library_construction_protocol: Total RNA was extracted from FACS sorted cells using a GeneJet kit (Thermo Scientific), and cDNA was synthesized using high fidelity Accuscript (Agilent) primed from a gene-specific primer (EBV reverse, annealing to the 3'UTR). Total RNA was extracted from FACS sorted cells using a GeneJet kit (Thermo Scientific), and cDNA was synthesized using high fidelity Accuscript (Agilent) primed from a gene-specific primer (EBV reverse, annealing to the 3'UTR). The S cDNA was PCR amplified as 3 separate fragments that together fully cover the RBD.  The PCR fragments were amplified for Illumina sequencing in two rounds.  In the first round, primer overhangs added sequences for annealing to the Illumina sequencing primers. In the second round, primer overhangs added unique barcodes and Illumina adaptors.  To determine the frequencies of mutations in the naive library, the library plasmid DNA was also used as a template for PCR amplification and Illumina sequencing. The S cDNA was PCR amplified as 3 separate fragments that together fully cover the RBD.  The PCR fragments were amplified for Illumina sequencing in two rounds.  In the first round, primer overhangs added sequences for annealing to the Illumina sequencing primers. In the second round, primer overhangs added unique barcodes and Illumina adaptors.  To determine the frequencies of mutations in the naive library, the library plasmid DNA was also used as a template for PCR amplification and Illumina sequencing.