Stimulation of reverse transcriptase generated cDNAs with specific indels by template RNA structure: retrotransposon, dNTP balance, RT-reagent usage

Penno, Christophe; Kumari, Romika; Baranov, Pavel V.; van Sinderen, Douwe; Atkins, John F.

TGIRT RT reactions involving template switching are described in Supplementary Methods. Analysis of the candidate retrotransposon slippage cassettes was performed using a specific DNA primer complementary to the 3 end segment of the test RNA. A mix of 100 ng RNA with 4 l 10 M specific primer and 10 l 2× TGIRT 'low salt' buffer in a total volume of 18 l, was incubated at 65 • C for 5 min and chilled on ice. Then 1 l 10 M TGIRT enzyme was added. The premix was pre-incubated at room temperature for 30 min. Reaction was initiated by adding substrate dNTPs as indicated in the text and incubated for 10 min at room temperature. In the final 20 l reaction mix, the final concentration of primer was 2 M and of TGIRT enzyme was 500 nM. Then, 1 l 5 M NaOH was added and incubated at 95 • C for 3 min. It was neutralized with 1 l 5 M HCl. cDNA was then purified with a silica-based column following the procedure described in Supplementary Methods. Elution was with 20 l RNase free water (Supplementary Table S1 ).