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Verification of snoRNA Transcription and 5'-ends

snoRNA transcription was assessed by reverse transcription of total RNA with oligonucleotide primers complementary to internal snoRNA sequence. Sequences of internal snoRNA primers are available by WWW [Lowe & Eddy, 1998]. Primer extension reactions were carried out using the same conditions as used for 1 mM [dNTP] rRNA primer extensions. RNA sequencing ladders were run adjacent to snoRNA primer extensions on 8% polyacrylamide gels to assess fragment lengths. Primer extensions on RNA from snoRNA-disrupted yeast strains were run next to primer extensions with wild-type strain RNA to verify loss of the snoRNA band of expected size.

Todd M. Lowe