GSM8779280: E14_mESC_Rep37_Seq2; Mus musculus; OTHER
Nuclei were isolated by incubating 1e6 cells in 1 mL ice-cold NE1 Buffer (20 mM HEPES, 10 mM KCl, 1 mM MgCl2, 0.1% Triton X-100, 20% glycerol, 1x Protease Inhibitor (Roche 4693132001)) for 5 min on ice. Nuclei were pelleted by centrifuging at 500 g for 5 min at 4C, and rinsed in 1 mL Buffer M (15 mM Tris-HCl pH 8.0, 15 mM NaCl, 60 mM KCl, 0.5 mM spermidine). Nuclei were re-pelleted by centrifuging at 500 g for 5 min at 4C, and were resuspended in 200 µL Buffer M with 1 mM SAM (NEB B9003S) and 10 µL high-concentration EcoGII (custom order from NEB). For footprinting, nuclei were incubated at 37C for a total of 30 min, with 1 µL 32 mM SAM stock spike-in after 15 min to supplement the reaction. Footprinted nuclei were pelleted by centrifuging at 500 g for 5 min at 4C, resuspended in 200 µL Buffer M with 1 mM CaCl2 and 0.02 U MNase (Sigma N3755), and incubated on ice for 30 min for MNase digest. 2 mM EGTA was added to stop the MNase reaction. For purifying DNA, samples were treated with 10 µL RNaseA (ThermoFisher AM2270) for 10 min at 37C, followed by the addition of 2 µL 10% SDS and 2 µL 20 mg/mL Proteinase K (ThermoFisher AM2548) and incubation >2 hr (up to overnight) at 65C. To extract DNA, an equal volume (~250 µL) phenol-chloroform-isoamyl alcohol was added, samples were mixed by brief vortexing, and samples were centrifuged at max speed for 2 min. The aqueous phase was transferred to a fresh tube, and 0.1x volumes 3M NaOAc, 2x volumes ice-cold 100% ethanol, and 1 µL GlycoBlue co-precipitant (Thermo AM9516) were added. Samples were mixed by inverting tubes and incubated overnight at -20C, centrifuged at max speed for 30 min at 4C, washed with 500 µL ice-cold 70% ethanol, air dried, and resuspended in 30 µL Buffer EB. DNA concentrations were measured by Qubit dsDNA High Sensitivity Quantification Kit (ThermoFisher Q32851). Up to 1 µg DNA was prepared into PacBio SMRT libraries with the SMRTbell Express Template Prep Kit 2.0 / 3.0 per manufacturer's directions. Briefly, this includes DNA damage repair, end repair, A-tailing, ligation of barcoded SMRTbell adapters, exonuclease clean-up, and 1x AMPure PB bead clean-up. DNA concentrations were measured by Qubit dsDNA High Sensitivity Quantification Kit and fragment size distributions were estimated with the Femto Pulse.