title: GSM5420836: M8_repA_PROseq; Homo sapiens; OTHER
library_construction_protocol: PRO-seq libraries were prepared as previously described (Mahat et al, 2016; Judd et al., 2020) with minor modifications. For each replicate timepoint, both floating cells in the growth medium and cells removed by scraping in 1x PBS were collected, pooled, and centrifuged at 1000xg for 5 min. Cells were resuspended in 1x PBS and 10% was removed for FACS analysis before completing the wash. 1mL of buffer P (10mM Tris-Cl pH 8.0, 10mM KCl, 250mM sucrose, 5mM MgCl2, 1mM EDTA, 0.05% Tween-20, 0.5mM DTT, 10% Glycerol) was used to gently resuspend cell pellets, before adding an additional 9mL of buffer P and incubating on a shaker for 5min. Permeabilization was assessed with trypan blue and samples not initially permeabilized were again incubated on a shaker with buffer P containing 0.05% NP-40 for 5min. Permeabilized cells were then centrifuged at 1000xg for 5min before resuspension in 1mL buffer F (50mM Tris-CL pH 8.0, 40% glycerol, 5mM MgCl2, 0.1 mM EDTA, 0.5mM DTT), transferred to a 1.5mL tube, and centrifuged at 1000xg for 5min. Finally, permeabilized cells were resuspended in 55µL of buffer F and 1µL of RNase-inhibitor was added before snap-freezing in liquid nitrogen and storage at -80°C.  0.9-4.5 x 106 permeabilized cells were mixed with permeabilized Drosophila S2 nuclei in all biotin-NTP run-ons (1 x 106 Dm nuclei in each A replicate and 5 x 104 in each B replicate). Run-on RNA was then base-hydrolyzed for 10min on ice and enriched with M280 streptavidin-beads. Full-length PRO-seq libraries were prepared as above with minor modifications. Nuclei were prepared in duplicate as above from asynchronous and 2hr mitotic-release cells, totaling ~1x107 nuclei per sample. Immediately following nuclei isolation, each sample was split into two separate run-on reactions. After nuclear run-on, TRIzol was added to stop the reaction and RNA was extracted. RNA was precipitated and resuspended in water, bound to streptavidin M280 beads and extensively washed. RNA was then eluted, extracted, bound to beads again, and washed. Nascent RNA was eluted from the beads and remaining ribosomal RNA was removed using riboZero Gold (Illumina). Samples split at the run-on step were then pooled and 0.01ng of total Drosophila RNA (rRNA-depleted) was added as a spike-in control. ATAC-seq was performed as previously described (Buenrostro, Giresi, et al, 2013) with minor modifications. 50,000 permeabilized cells were pelleted and then incubated for 30min in ATAC-seq reaction buffer at 37°C in a shaker at 1000rpm. DNA was then extracted with phenol-chloroform before size selection with AMPure beads. PRO-seq libraries were prepared as previously described (Mahat et al, 2016; Judd et al., 2020) with minor modifications. Base-hydrolyzed run-on RNA underwent 3'-adapter ligation and a second bead binding. Both end-repair reactions and the 5'-ligation were all performed with nascent RNA still bound to the beads. These on-bead reactions were done in a total volume of 20µL with constant rotation before elution from the beads and the subsequent reverse transcription and PCR steps. Test amplifications were performed on 5% of the library and samples were amplified the ideal number of cycles. Following final amplification, libraries were PAGE-purified to remove adapter-dimers and select molecules below 650bp in size. Libraries were then sequenced on an Illumina NextSeq 550, producing single-end 75bp reads. Full-length PRO-seq libraries were constructed using the Illumina TRU-seq stranded low-input RNA-seq kit following the standard protocol and then sequenced on an Illumina NextSeq 550, producing paired-end 150bp reads. ATAC-seq libraries were test amplified using 5% of the sample following AMPure clean-up. Samples were amplified the ideal number of cycles and libraries were then sequenced on an Illumina NextSeq 550, producing single-end 75bp reads.