Yeast mitochondrial RNA polymerase in vitro reactions with DNA oligonucleotide AF-EG-65 input 20 minute timepoint
Samples were generated using in vitro reactions with recombinantly expressed and purified preparations of yeast mitochondrial RNA polymerase with a 27nt DNA oligonucleotide as input. This sample was the 20 minute timepoint. We found that Rpo41 generated DNA->RNA covalently linked extension products on the DNA oligonucleotide. Samples were purified using phenol:chloroform extraction and ethanol precipitation. The resulting purified nucleic acid was then prepared into libraries using DruSeq, a derivitized Illumina TruSeq Small RNA protocol using in-house adapters and RT primers that includes addition of polyethylene glycol in the adapter ligation steps. Reverse transcription was performed using SuperScript II. Libraries were amplified using 16 rounds of PCR and gel purified using Qiagen gel purification kit according to manufacturer's instructions. These data were NOT adapter-trimmed prior to upload.