GSM9380631: mESCs, Control, 6min, rep1; Mus musculus; MNase-Seq
After harvesting serum-grown mESCs, cells were washed in cold 1x phosphate buffered saline solution (PBS). Cell number and viability were measured with CountessTM 3 Automated Cell Counter (Invitrogen). MNase-seq digestions proceeded with 1 x 10^6 mESCs. mESC samples were centrifuged at 2000 rpm for 5 minutes to remove 1x PBS. Cells were resuspended in 1 mL cold Nuclear Extraction Buffer (20 mM HEPES pH 8.0, 10 mM KCl, 0.1% Triton, 20% glycerol, 0.5 mM spermidine) and incubated on ice for 10 minutes to facilitate nuclei isolation. Nuclei were centrifuged at 2500 rpm for 10 min, then resuspended in 40 uL Reaction Buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM spermidine, 0.01% digitonin, 2 mM EGTA, 10 mM CaCl2, 10 mM MgCl2). Samples were incubated at 37°C for 5 minutes prior to starting digestion to allow for reaction temperature equilibration. 0.07 U micrococcal nuclease (Sigma) was added to each sample along with nuclease-free water (Invitrogen) to adjust reaction volumes to 50 uL. Reactions proceeded at 37°C for time points of 3, 6, 7, 9, 11, 12, and 15 minutes. At each time point, 10 uL of reaction (0.2 x 106 mESC nuclei) was removed and quenched with 10 uL 2x Stop Buffer (500 mM NaCl, 10 mM EGTA, 0.1% digitonin, 1% SDS, 100 ug RNase A, 50 ug glycogen). Digested DNA was purified by phenol:chloroform:isoamyl alcohol extraction protocol. Purifed DNA concentration and sample quality was determined via Qubit 4 fluorometer and Agilent D1000 Tapestation respectively. Sequencing libraries were prepared using the SRSLY PicoPlus NGS Library Prep Kit and protocol, described as follows. 10 ng of digested DNA was combined with SRSLY ssEnhancer and incubated on ice for 2 minutes, followed by heat shock at 98C for 3 minutes and another incubation on ice for 2 minutes. Phosphorylation and ligation reaction was achieved by mixing sample with SRSLY Adapter A, SRSLY Adapter B, and SRSLY Master Mix and then incubating samples at 37C for 1 hour. Samples were then purified in a final volume of 15 uL nuclease-free water via 0.54x AMPure bead clean to enrich for small fragments. To each sample, SRSLY PCR Master Mix and unique P5 and P7 barcoded primers were added. The following PCR protocol was used: 98C for 3 minutes, 98C for 20 seconds, 65C for 30 seconds, 72C for 30 seconds, 72C for 1 minute with steps 2-4 repeated for 10 cycles. Final library purification was achieved via 0.95x AMPure bead clean, after which library quality was assessed via Agilent TapeStation.