title: GSM8855116: UMI-4C in MCF-7 cells after control GapmeR uptake, replicate 1; Homo sapiens; OTHER
library_construction_protocol: Metabolic labeling and SLAM-seq were then performed as previously described (Herzog et al., 2017; Zuckerman et al., 2020), with minor modifications. All steps were performed in the dark until the chemical conversion with iodoacetamide (IAA). After 48h hours, the growth medium was replaced with fresh media supplemented with 500 µM 4-Thiouridine (4sU, Sigma), and replaced again with fresh 4sU after every time point. In total, we collected RNA from cells at time 0 (unlabeled), and after 4, 8 and 24h hours post-4sU addition. RNA extraction was carried out as described above with the addition of DTT (0.1 mM final concentration) to keep the samples under reducing conditions, and IAA conversion was performed as described previously (Zuckerman et al., 2020). UMI-4C was performed as previously described (Schwartzman et al., 2016) CUT&RUN reactions were performed as previously described (Meers et al., 2019). RNA-seq libraries were prepared using the CORALL mRNA-Seq Kit V2 (Lexogen), following the manufacturer's instructions. SLAM-seq libraries were prepared using the NEBNext Ultra II Directional Library Kit (NEB), according to the manufacturer's protocol. SLAM-seq libraries were generated as described before (Schwartzman et al., 2016). CUT&RUN libraries were prepared as described previously (Meers et al., 2019).