GSM7033898: 515_AMlib3_1511A_IP; Saccharomyces cerevisiae; OTHER
RNA extraction was performed from yeast pellets using Acid Phenol:Chloroform pH 4.5 and Tris-EDTA-SDS (TES) buffer (0.01 M Tris-HCl pH 7.5, 0.01 M EDTA, 0.5% w/v SDS). Samples were further treated with rDNase (cat no 740.963, Macherey-Nagel) and column purified (cat no 740.948, Macherey-Nagel).  m6A levels were determined by m6A-seq2, as previously described {Dierks, 2021 #308; Schwartz, 2013 #4}.Briefly, RNA was poly-A selected twice using Dynabeads mRNA DIRECT kit, and fragmented to ~150 bp fragments. 3' RNA barcode adaptors were then ligated (see Table S6), and all samples were pooled for m6A-IP-based enrichment. 90% of the mRNA material was subjected to two rounds of immunoprecipitation using two distinct anti-m6A antibodies, as follows: The RNA was first incubated for four hours with protein G beads (invitrogen) coupled to a Synaptic Systems poly-clonal m6A antibody (cat. 7945). Next, it was incubated overnight with protein A beads (invitrogen) coupled to a Cell Signaling poly-clonal m6A antibody (cat. D9D9W). RNA from input and IP libraries were reverse transcribed and PCR amplified as previously described  {Dierks, 2021 #308}. cDNA libraries were quantified using Qubit RNA HS kit (Life technologies) and Tapestation High Sensitivity D1000 ScreenTape (Agilent Technologies), pooled with a ratio of ⅓ Input to ⅔ IP samples, and sequenced.