Reverse Transcription with Gel Purification

RT was performed with gel purified RT primers 5′-pGG-B-AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT-SP18-CTCGGCATTCCTGCTGAACCGCTCTTCCGATCT-CCTTGGCACCCGAGAATTCCA-3′, where B indicates a 5-nt barcode of sequence ATCAC, CGATG, TAGCT, GCTCC, ACAGT, CAGAT, TCCCG, GGCTA, AGTCA, CTTGT, TGAAT or GTAGA. RT products were detected by incorporating α-³²P-dCTP in the reaction. RT products intended for circularization were gel purified. For the data in Figures 4 and 5, we eluted the cDNA from crushed gel pieces in 300 mM NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA) during an overnight incubation at room temperature with constant rotation; eluted material was ethanol precipitated before circularization. We have since modified our approach to increase elution yield by eluting in TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0) and incubating at 37°C overnight with constant rotation. With this buffer, we can concentrate the eluate (either by butanol extraction or SpeedVac) before precipitating the sample in a single tube.