GoKawiilSAMOSA from E14 mouse ES cells
We compiled a large compendium of SAMOSA data from wild-type E14 mouse ES cells, comprising both newly generated datasets and published datasets from our laboratory, including: (1) our study on the function of ISWI-family remodelling complexes3; (2) samples prepared with our SMRT-Tag protocol, which uses a transposase-based strategy for PacBio library preparation from lower input amounts of footprinted DNA38; and (3) mouse ES cells labelled with the halogenated thymidine analogue BrdU for varying time points in our study of single-fibre accessibility patterns in newly replicated chromatin52. Only samples with a mononucleosome–dinucleosome ratio (MDR; calculation detailed in ‘Data analysis and visualization’) greater than 10 were included for analysis. All cell lines were routinely tested for mycoplasma contamination. No statistical models were used to predetermine sample size. Neither blinding nor randomization was used.
Cell culture
For newly generated libraries, E14 mouse ES cells were grown on gelatin-coated (0.1% solution in 1× PBS) tissue culture plates. Cells were cultured in mouse ES cell medium (DMEM with GlutaMAX (Thermo Fisher Scientific, 10566-016)), supplemented with 15% fetal bovine serum (FBS; Thermo Fisher Scientific, SH30071.03), 14.2 mM 2-mercaptoethanol (Bio-Rad, 1610710), 1× NEAA (Thermo Fisher Scientific, 11140-50), 1 mM sodium pyruvate (Thermo Fisher Scientific, 11360-070) and 1× LIF (purified by the laboratory of B. Panning). Some samples were cultured with MEK (1 µM PD0325901 (Apex Bio/Fischer, A3013-25)) and GSK3β (3 µM CHIR99021 (Apex Bio/Fischer, A3011-100)) inhibitors (2i). The medium was changed daily and cells were passaged with 1× TrypLE (Thermo Fisher Scientific, 12605010) when confluent. To collect mouse ES cells, cells were rinsed with 1× PBS (Thermo Fisher Scientific, 10010023), dissociated with 1× TrypLE, quenched with mouse ES cell medium and centrifuged at 500g for 5 min. All mouse ES cell lines used in this study were regularly tested for mycoplasma contamination (Lonza LT07-318).
MTase footprinting and library preparation
Live cell counts were estimated (Countess 3) to ensure equal numbers of nuclei per unit MTase were used in each footprinting reaction. Nuclei were isolated by incubating 1 × 106 live mouse ES cells in 1 ml ice-cold NE1 buffer (20 mM HEPES, 10 mM KCl, 1 mM MgCl 2 , 0.1% Triton X-100, 20% glycerol and 1× protease inhibitor (Roche, 4693132001)) on ice for 5 min. Nuclei were pelleted by centrifugation at 500g for 5 min at 4 °C. Nuclei were rinsed in 1 ml buffer M (15 mM Tris-HCl pH 8.0, 15 mM NaCl, 60 mM KCl and 0.5 mM spermidine) and re-pelleted by centrifugation at 500g for 5 min at 4 °C. For footprinting, nuclei were resuspended in 200 µl buffer M with 1 mM SAM (NEB, B9003S) and 10 µl high-concentration EcoGII (custom order from NEB). Nuclei were incubated at 37 °C for 30 min, with a spike-in of 1 µl 32 mM SAM stock halfway through the footprinting reaction. For light MNase digest to liberate chromatin fragments, footprinted nuclei were pelleted by centrifugation at 500g for 5 min at 4 °C, resuspended in 200 µl buffer M with 1 mM CaCl 2 and 0.02 U MNase (Sigma N3755) and incubated on ice for 30 min. To quench the MNase reaction, 2 mM EGTA was added.
To purify DNA, samples were treated with 10 µl RNaseA (Thermo Fisher Scientific, AM2270) for 10 min at 37 °C, followed by the addition of 2 µl 10% SDS and 2 µl 20 mg ml−1 proteinase K (Thermo Fisher Scientific, AM2548) for more than two hours (up to overnight) at 65 °C. To extract DNA, an equal volume (around 250 µl) phenol-chloroform-isoamyl alcohol was added, and samples were mixed by brief vortexing and centrifuged at maximum speed for 2 min. The aqueous phase was transferred to a fresh tube along with 0.1 volumes 3 M NaOAc, 2 volumes ice-cold 100% ethanol and 1 µl GlycoBlue co-precipitant (Thermo Fisher Scientific, AM9516). Samples were mixed by inverting tubes and incubated overnight at −20 °C, centrifuged at maximum speed for 30 min at 4 °C, washed in 500 µl ice-cold 70% ethanol, air dried and resuspended in 30 µl buffer EB. DNA concentrations were measured using the Qubit dsDNA High Sensitivity Quantification Kit (Thermo Fisher Scientific, Q32851). Up to 1 µg DNA was prepared into PacBio SMRT libraries with the SMRTbell Express Template Prep Kit 2.0 as per the manufacturer’s directions. Fragment size distributions were estimated by Femto Pulse (Agilent). Libraries were sequenced on PacBio Sequel II 8M SMRT cells.
SAMOSA from SOX2-FKBP mouse ES cells
Degron-tagged Sox2FKBP-F36V (hereafter referred to as SOX2-FKBP) mouse ES cells were provided by the laboratory of E. de Wit60. SOX2-FKBP mouse ES cells were cultured in the same mouse ES cell medium described above (including 2i) and were plated at a density of 5 × 105 per well in 6-well plates 24 h before collection. For degradation, SOX2-FKBP mouse ES cells were treated with 0.5 µM dTAGV-1 (Tocris 6914) for varying time points (30 min, 2 h or 6 h). As a control, SOX2-FKBP mouse ES cell samples were treated with 0.5 µM dTAGV-1-NEG (Tocris 6915) for six hours before collection. All experiments were performed in biological duplicate. Footprinting and downstream steps were performed as detailed in the section above, except that instead of the MNase digest step, we sheared purified genomic DNA with Covaris g-TUBEs (Covaris, 520079; 4,600g for six passes).
Western blot for SOX2 protein in nuclear extracts
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