title: GSM4420183: ACE2 library sorted for low binding to the RBD of S protein from SARS-CoV-2 (Replicate 1; PCR fragments C, D and E); Homo sapiens; 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 one of two gene-specific primers. The ACE2 cDNA was PCR amplified as five separate fragments that together fully cover the diversified regions of ACE2 present in the SSM library. 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. Gene-specific primers for reverse transcription with Accuscript were: ACE2_RevTrans_411R (CAGTACTGTAGATGGTGCTC) for fragments A and B, and ACE2_RevTrans_1685R (CAGTTTCTGTCCAGCTTCTG) for fragments C, D and E. Primer pair for 1st round of PCR (Fragment A): MiSeq_CCR5_myctag_F (TCTTTCCCTACACGACGCTCTTCCGATCTCGAGCAAAAGCTGATCTCAG) and Illumina_ACE2_286_rev (GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTTTGGGCAAGTGTGGACTGT) Primer pair for 1st round of PCR (Fragment B): Illumina_ACE2_147_for (TCTTTCCCTACACGACGCTCTTCCGATCTAGCCGAAGACCTGTTCTATC) and Illumina_ACE2_354_rev (GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTTCTGAGAGCACTGAAGAC) Primer pair for 1st round of PCR (Fragment C): Illumina_ACE2_827_for (TCTTTCCCTACACGACGCTCTTCCGATCtccctgctcatttgcttggt) and Illumina_ACE2_1044_rev (GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGAACATTTCCTGGGTCC) Primer pair for 1st round of PCR (Fragment D): Illumina_ACE2_1044_for (TCTTTCCCTACACGACGCTCTTCCGATCTggacccaggaaatgttcaga) and Illumina_ACE2_1270_rev (GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTTAGGTGTGGCTGCAGAAAG) Primer pair for 1st round of PCR (Fragment E): Illumina_ACE2_1304_for (TCTTTCCCTACACGACGCTCTTCCGATCttggtcttctgtcacccgat) and Illumina_ACE2_1621_rev (GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGCTGCTTGACAAAGTGCTTC) Primer pair for 2nd round of PCR (added I5 and I7 Illumina adaptors): MiSeq_Start_Adaptamer (AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT) and MiSeq_Index_Adaptamer (CAAGCAGAAGACGGCATACGAGAT-6nt-barcode-GTGACTGGAGTTCAGACGTGTGCTCTTC) Amplicons were deep sequenced on a NovaSeq 6000 using a 2x250 nt paired end protocol.
attributes.tag: GEO Accession
attributes.value: GSM4420183