title: GSM5241226: ChIP-seq-D-Pol2-SPT5AID7-shNEDD4-Vehicle; Homo sapiens; ChIP-Seq
library_construction_protocol: For ChIP-seq in human DLD1 cells, 20-50 million cells were crosslinked with 1% paraformaldehyde (Electron Microscopy Sciences) in PBS for 10 min at r.t. Spike-in mouse embryonic fibroblast fixed cells were added to samples as described previously (Orlando et al., 2014). Chromatin was sonicated with the Covaris E220 for 4 min with 10% duty cycle, 140 peak intensity power, 200 cycles per burst. Immunoprecipitations were carried out with antibodies and Dynabeads Protein G (Invitrogen). For ChIP-seq in yeast cells, cells were fixed with 2.2% formaldehyde for 15 min. PRO-seq sample preparation was performed according to the previously described qPRO-seq protocol (Judd et al., 2020). ~5 million DLD-1 nuclei mixed with spike-in Drosophila S2 nuclei were used for nuclear run-on assay. For pulse-chase nascdnt RNA labeling with TT-seq, cells were treated with 250 nM NVP-2 for 1 h, followed by nascent RNA labeling with 500 µM 4-thiouridin (Sigma # T4509) for 15 min. For ChIP-seq, DNA libraries were prepared by the HTP Library Preparation Kit for Illimina (KAPA). For PRO-seq, adapter sequences previously described (Mahat et al., 2016) were used. PRO-seq libraries were prepared according to the previously described qPRO-seq protocol (Judd et al., 2020). TT-seq library was prepared as described previously (Schwalb et al., 2016; Rosencrance wt al., 2020).