GSM4090946: Nab3 rrp6∆ rep3; Saccharomyces cerevisiae; OTHER
Defrosted lysates were treated for one hour at 18°C with DNase I to solubilize the chromatin, clarified by centrifugation, followed by stringent, denaturing, two-step affinity purification of the tagged protein as described in the original protocol (Bohnsack et al., 2012) with minor changes. Bound RNA partially degraded with RNase, was ligated to modified 5' and 3' linkers adapted to sequence RNA molecules from the 3' end. Covalent RNA-protein complexes were isolated by Gel Elution Liquid Fraction Entrapment Electrophoresis (Gelfree). Following Proteinase K digestion of the bound proteins, RNA fragments were amplified by RT-PCR. Between half to ¾ of the cDNA solution was used to performed multiple PCR reactions in a final volume of 25 µL using the following conditions: 0.4 µM of each primers, 0.2 mM dNTP, 2.5 U LA Taq DNA polymerase from Takara, 1X LA PCR Buffer II and 2 µL of cDNA per reaction with the programme: 95°C 2', 95°C 30'', 58°C 45'', 72°C 1', 72°C 5'. PCR were pooled and treated with 200 U of Exonuclease I (NEB) per milliliter of PCR reaction for 1 hour at 37°C. After Exonuclease I inactivation for 20' at 80°C, DNA was purified on PCR clean up columns (NucleoSpin® Gel and PCR Clean-up, Macherey-Nagel) and sent for sequencing using Illumina technology (NextSeq sequencing).
GEO Accession
GSM4090946