GSM4419115: TAPBPR 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 TAPBPR 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_TAPBPR_273_for (TCTTTCCCTACACGACGCTCTTCCGATcttgctccatgctgactgca) and Illumina_TAPBPR_483_rev (GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTCAGTGGCAAGTTCAGCGTTG) Primer pair for 1st round of PCR (Fragment B): Illumina_TAPBPR_466_for (TCTTTCCCTACACGACGCTCTTCCGATCTacgctgaacttgccactgag) and Illumina_TAPBPR_671_rev (GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGTCCAGCTGTACACCAACTG) Primer pair for 1st round of PCR (Fragment C): Illumina_TAPBPR_649_for (TCTTTCCCTACACGACGCTCTTCCGATCTggtcagttggtgtacagctg) and Illumina_TAPBPR_920_rev (GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTCCAGCAATGTCGCAGATGAG) Primer pair for 1st round of PCR (Fragment D): Illumina_TAPBPR_875_for (TCTTTCCCTACACGACGCTCTTCCGATCtggcaaacgaagctctgctg) and Illumina_TAPBPR_1148_rev (GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGGGACAACCTGGGTGCTG) 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.
GEO Accession
GSM4419115