title: K562_InVitroPolyA_Ribo0
design: CELL CULTURE: K562-MycER cells - from here on named K562 - were grown and maintained in RPMI (Invitrogen Corporation; Cat No. 23400-021) containing 5% FBS in a 5 %C02 incubator at 37C. METABOLIC LABELING: K562 cells were treated with 500uM of 5-Ethynyl Uridine (Jena Bioscience CLK-N002-10) for 60 minutes. RNA EXTRACTION: Total RNA was extracted using Qiazol (Qiagen 79306). rRNA DEPLETION: 10ug of Total RNA from K562 cells were Ribo depleted using RiboMinus Eukaryote Kit (Thermofisher Scientific A1083708). 2 rounds of Ribo depletion were performed. POLYADENYLATION: 1ug of Ribo depleted RNA was denatured, and Poly(A) tailed in vitro using E.coli Polymerase NEB (NEB# M0276); RNA was incubated 30 minutes at 37C. RNA was cleaned using RNAClean XP beads (Beckman Coulter) and then loaded into MACS columns for mRNA purification. LIBRARY PREPARATION: 100ng of mRNA were used as an input for Nanopore library preparation.