We amplified the cDNA and genomic DNA samples using GoTaq Flexi DNA polymerase (Promega Corporation, www.promega.com). PCR products were purified using ExoSap-IT (USB Corporation, www.usbweb.com) followed by SNaPshot to extend primer by a single fluorescently labeled ddNTPs. Fluorescently labeled products were purified using calf intestinal phosphatase (CIP, New England BioLabs, www.neb.com) and separated by capillary electrophoresis on ABI3130 (Applied Biosystems). Quantification was done using GeneMapper v4.0 software (Applied Biosystems), and transcript abundance was measured by peak intensities associated with each allele. Ratio of B and D allele in both cDNA and gDNA pools was computed, and t-test (one tail, unequal variance) was done to validate expression difference and polarity of parental alleles.
