GSM3690822: PDGF-B library sorted for high binding to 30 nM PDGFRbeta-Fc; Homo sapiens; OTHER
Plasmid DNA was harvested from unsorted and sorted cells.  The PDGF-B insert was amplified for deep sequencing in two PCR 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 (Fragment A): PDGF_FragA_DeepSeq_For (TCTTTCCCTACACGACGCTCTTCCGATCTCATATGGGAGGGAGTTCTGC) and PDGF_FragA_DeepSeq_Rev (GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTTCTGTTGTTACAGCAACCGC). Primer pair for 1st round of PCR (Fragment B): PDGF_FragB_DeepSeq_For (TCTTTCCCTACACGACGCTCTTCCGATCTGCGTGGAAGTTCAACGCTG) and PDGF_FragB_DeepSeq_Rev (GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTTAAGCTCGAGACCGCCGCTA). 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 HiSeq 2500 using a 2x250 nt paired end protocol.