GSM4824311: Tapasin library sorted for rescue of surface HLA-A2 expression in tapasin-KO cells (Replicate 2); Homo sapiens; OTHER
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.  The tapasin cDNA was PCR amplified as four overlapping fragments for full coverage by Illumina sequencing.  In a first round of PCR, primer overhangs added sequences for annealing to the Illumina sequencing primers. In a second round of PCR, primer overhangs added unique barcodes and Illumina adaptors.  To determine the frequencies of mutations in the naive library, the library plasmid DNA was used as the template for PCR amplification and Illumina sequencing. Primer pair for 1st round of PCR (Fragment A): Illumina_tapasin_5F (TCTTTCCCTACACGACGCTCTTCCGATCTGCCGTAATCGAGTGTTGGT) and Illumina_tapasin_235R (GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTAAGGAACGAACCGACTCATC) Primer pair for 1st round of PCR (Fragment B): Illumina_tapasin_183F (TCTTTCCCTACACGACGCTCTTCCGATCTGTACCCAAGAGGTGCTCCA) and Illumina_tapasin_405R (GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTCGCCATTGTTATCAGTACGG) Primer pair for 1st round of PCR (Fragment C): Illumina_tapasin_386F (TCTTTCCCTACACGACGCTCTTCCGATCTCCGTACTGATAACAATGGCG) and Illumina_tapasin_602R (GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGTAGCAGCCAACAGCAAATG) Primer pair for 1st round of PCR (Fragment D): Illumina_tapasin_630F (TCTTTCCCTACACGACGCTCTTCCGATCTAGCACAAGAGGGAGCCGTA) and Illumina_tapasin_876R (GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTTCAGGTGGAGCTTCGCCA) Primer pair for 1st round of PCR (Fragment E): Illumina_tapasin_858F (TCTTTCCCTACACGACGCTCTTCCGATCTGGCGAAGCTCCACCTGAA) and Illumina_tapasin_1040R (GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGCAAATGCCCTGACAGTGA) Primer pair for 1st round of PCR (Fragment F): Illumina_tapasin_951F (TCTTTCCCTACACGACGCTCTTCCGATCTGGTCGATCTCAGAAAGCTG) and Illumina_tapasin_1135R (GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTTACCTCCGCTGATCTTCCT) 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.