GSM7623265: Slr1p IP, AB2756, biol repl 1; Schizosaccharomyces pombe; RIP-Seq
1 μg/μl final concentration of proteinase K (Invitrogen) solution and 0.125% final concentration of SDS solution (Bioshop) was added to immunoprecipitated RNA. Samples were then incubated at 37°C for 1 hour to digest immunoprecipitated proteins first, and 65°C for another hour to reverse crosslinking. This was followed by phenol-chloroform extraction of RNA and ethanol precipitation. Equal volumes of acidic phenol pH 4.3 (Fisher) was added to each sample which was vortexed to mix and centrifuged at 21000xg for 10 minutes at 4°C. The top aqueous layer was removed to a new tube to which equal volumes of Phenol/Chloroform/Isoamyl Alcohol (25:24:1 mixture, Fisher) was added following by vortexing and centrifugation at 21000xg for 10 minutes at 4°C. The top aqueous layer was removed to a new tube again and to this, equal volumes of Chloroform/Isoamyl Alcohol (24:1 mixture, Sigma) was added. The centrifugation step above was repeated and the top layer was removed to a new tube again. Finally, 20 μg glycogen (Sigma), 1/10th of the volume 3M NaOAc pH 5.2 (Sigma) and 1 mL ice-cold 100% absolute ethanol (Greenfield Global) was added to each RNA sample which was precipitated next at -80°C until sequencing. Input RNA samples were extracted using the hot phenol RNA extraction method and precipitated as described above until sequencing. The NEBNext Ultra II Directional RNA Library Prep Kit for Illumina was used for library preparations following manufacturer's protocol.