NextSeq 500 sequencing; In-vivo and in-vitro RIP-seq of WT and Delta NLS1/2 mutant of Xrn1
In-vivo and in-vitro RIP-seq of WT and Delta NLS1/2 mutant of Xrn1
In vivo or in vitro mRNA interactome profiles of FLAG-tagged WT and Delta NLS1/2 mutant were determined, each condition in duplicate, as follows. Condition I: in vitro. Purified Xrn1 samples were reacted in vitro with affinity purified poly(A)+ cellular mRNAs (purified using oligo(dT)20 beads) followed by RIP-seq. Condition II: in vivo. Cells were UV crosslinked in-vivo using Vari-X-Link UV-crosslinker. IP was done in Tandem with anti-FLAG magnetic beads followed by His purification with Ni-NTA resin under denaturing condition. RNA was released by incubating the bead conjugated RNA-protein complex with Proteinase K at a final concentration of 100µg/ml for 1 h at 56°C. Cells were grown in YPD medium for 7 generations to reach mid-log phase. WT cells and Xrn1 NLS mutant cells were grown for the experiment. Total RNA was purified with standard Phenol: Chloroform extraction Library construction was done using PAS sequencing protocol (Yoon et.al. 2021, Methods in Enzymology)