GSM4296327: PRO-seq-14-NELFC-AID-FP-Aux; Homo sapiens; OTHER
For ChIP-seq, 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) for NELF-C, NELF-E, SPT5, Pol II CTD Ser2P, Pol II CTD Ser5P, XRN2, DCP2 ChIP-seq in NELF-C-AID cells. 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 Protein A/G-agarose beads (Santa Cruz) or Dynabeads Protein G (Invitrogen).  PRO-seq sample preparation was performed according to the previously published protocol (Mahat et al., 2016b) with minor modifications. 10 million DLD-1 nuclei were mixed with spike-in Drosophila S2 nuclei. Nuclear run-on assay was performed with 4 biotinylated nucleotides (PerkinElmer). 5' cap was removed by RppH (NEB).  PRO-cap libraries were prepared as described previously (Kwak et al., 2013; Mahat et al., 2016b) with minor modifications. NELF-C-AID cells were treated with or without 500 µM auxin for 2 h, followed by nuclear extraction. ~10 million NELF-C-AID nuclei with 0.5 million spike-in drosophila S2 nuclei were subjected to nuclear run-on reaction. For ChIP-seq, DNA libraries were prepared by the HTP Library Preparation Kit for Illimina (KAPA) and sequenced on the NextSeq 500 or NovaSeq 6000 (Illumina). For PRO-seq, adapter sequences previously described (Mahat et al., 2016) were used. DNA Libraries were size selected by PippinHT (Sage) or AMpure XP (Beckman Coulter) and sequenced on a NextSeq 500 (Illumina). For PRO-cap, PRO-seq adapter sequences were used instead of PRO-cap adapters. Libraries were then sequenced by NextSeq 500 in paired-end sequencing mode.