title: Sanger sequencing HPV70 and BCL11B exon 4 Homo sapiens: adult female cervix carcinoma
design: To detect the HPV70-BCL11B fusion transcripts, primers were designed to flank each side of the fusion splice junction from the HPV70 E1 5ss at genome position 943 to the BCL11B exon 4 3ss at GRCh37/hg19 genome position chr14:99642532 obtained from RNAseq split reads.  The HPV70 primer was 5-GAAGAACCACAGCGTCACAA and the BCL11B exon 4 primer was 5-TGCAAATGTAGCTGGAAGGC with a 214 bp amplicon.  First-strand cDNA synthesis was performed on 5 g of total RNA from either fresh tumor tissue or archival FFPE CIN3 tissue using the exon 4 primer and SuperScriptII RT (Thermo Fisher Scientific) following manufactures protocol.  The subsequent PCR to detect the presence of the fusion transcripts was performed using 35 cycles of 95C for 15 sec, 60C for 30 sec, and 72C for 45 sec. The resultant PCR products were resolved on 0.8% or 2.0% agarose gels, respectively, and the bands were excised using the Monarch DNA Gel Extraction Kit (New England Biolabs) for Sanger sequencing analysis.
library_name: 9918_595429_G10_034