GSM3593594: Wild type TAPBPR-tapasin chimera: Control for sequencing errors; Homo sapiens; OTHER
For yeast display, plasmid DNA was harvested from unsorted and sorted cells. For experiments in human Expi293F tapasin-KO cells, cDNA was synthesized from total RNA of the sorted cells and compared to the naive plasmid library.  In all cases, the scoop loop region was PCR amplified in 2 steps; in the first, primer overhangs added sequences for annealing to the Illumina sequencing primers, and in the second, primer overhangs added unique barcodes and Illumina adaptors. Primer pair for 1st round of PCR to amplify TAPBPR scoop loop: Illumina_TAPBPR_111F (GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTAGACTGTTTCCTGGTGAAGG) and Illumina_TAPBPR_229R (TCTTTCCCTACACGACGCTCTTCCGATCTCCTTGGAAATCGGTGAAGTC). Primer pair to amplify tapasin exon 2: Tapasin_Illumina_FOR (TCTTTCCCTACACGACGCTCTTCCGATCTGGCGACCGCCGTCTCAG) and Tapasin_Illumina_REV (GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTCAACAGCAGTGCACCGGGTC). 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 MiSeq using a 2x150nt PE v2 protocol (HLA-A2 processing experiments), Miseq using a 2x150nt PE NANO v2 protocol (validation of tapasin-kock out line), or HiSeq 2500 using a 2x250 nt paired end protocol.
GEO Accession
GSM3593594