GSM7233840: Sfp1_wt_biol_rep_1; Saccharomyces cerevisiae; OTHER
Cell extracts were prepared essentially as previously described (van Nues et al 2017) except that the frozen cells were lysed by pulverising the liquid-nitrogen-frozen cells in a mixer mill. Extract was subjected to tandem affinity purification, using IgG-sepharose beads followed by mild Rnase treatment, terminateion by 6M GuHCl followed by NTA beads purification; Dephosphorylate and add 3' adapter on beads; 5' phosphorylate using radioactive ATP; On-Bead 5' Adapter ligation; Elute Sfp-23P-RNA and run on PAGE; Eloctrotransfer onto NC membrane and cut out the radioactive band followed by proteinase K and phenol:chloroform extraction; Reverse transcribe and PCR amplify. See Haag et al. 2017