GSM4101201: UL74-Low Replicate1; Homo sapiens; OTHER
Total RNA was harvested from sorted cells using GeneJet RNA Purification Kit (Thermo Scientific) and cDNA prepared using Accuscript Hi-Fi (Agilent Genomics) primed with a gene-specific primer (RevTrans_PDGFRA_992R: TCATGCAGGTTGACAGCTTC). The D2-D3 encoding region 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): Illumina_PDGFRA_325_for (TCTTTCCCTACACGACGCTCTTCCGATCTGAGAATGAGCTTGAAGGCAG) and Illumina_PDGFRA_567_rev (GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTACAGATATAGGGCCCTACAG). Primer pair for 1st round of PCR (Fragment B): Illumina_PDGFRA_527_for (TCTTTCCCTACACGACGCTCTTCCGATCTAGGGCTTTAATGGGACCTTC) and Illumina_PDGFRA_770_rev (GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTCCTTTGCCTTTCACTTCTCC). Primer pair for 1st round of PCR (Fragment C): Illumina_PDGFRA_724_for (TCTTTCCCTACACGACGCTCTTCCGATCTGTGGTTGACCTTCAATGGAC) and Illumina_PDGFRA_983_rev (GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTTGACAGCTTCCAACTGGCT). 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
GSM4101201