GSM6562874: Rpa2-HTP WT_2; Schizosaccharomyces pombe; OTHER
Crosslinked samples were lyzed in TNMC100 (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1% NP-40, 5 mM MgCl2, 10 mM CaCl2, 5 mM β-mercaptoethanol, 50U of DNase RQ1 and a protease-inhibitor cocktail) with zirconia beads in a 50 mL conical. Cleared lysates were incubated with IgG Sepharose and washed three times with TMN600 (50 mM Tris-HCl pH 7.5, 600 mM NaCl, 0.1% NP-40, 1.5 mM MgCl2) and two times TMN100 (50 mM Tris-HCl pH 7.5, 100 mM NaCl, 0.1% NP-40, 5 mM MgCl2). Protein:RNA complexes were eluted with HaloTEV and the supernantant was subjected to nickel affinity purification in denaturing conditions. After extensive washing of the nickel beads, the addition of 3' linkers, 5' end radiolabeling, and 5' linker ligation was performed on beads. Protein:RNA complexes were then eluted, precipitated, separated on gels, and transferred to nitrocellulose membranes. After detection of labeled RNAs by autoradiography, the appropriate region was excised and treated with Proteinase K. The RNA was then isolated, reverse transcribed and PCR amplified as described  (Turowki et al., 2020). Following gel extraction of PCR amplicons corresponding to 140-200 bp, libraries were measured by Qbit and analyzed by Illumina sequencing. CRAC (Cross-linking and Analysis of cDNA)