GSM9219500: mES-E14-WT_1; Mus musculus; MNase-Seq
Aliquots of 1 x 107 cells were fixed 10 ml media with Formaldehyde, 37% (vol/vol) (Sigma, 47608-250ML) at a final of concentration 2% for 10 minutes at room temperature. This reaction was quenched with 1 M glycine to a final concentration 130 mM and centrifuged (5 min, 300 g). The supernatant was removed, and the cell pellet washed with cold PBS and reconstituted in 1 ml PBS. Cells were permeabilised with Digitonin (Sigma D-141; final concentration of 0.005% wt/vol). Cells were snap frozen and stored at -80 °C. Subsequently, defrosted, permeabilised cells were centrifuged (5 min, 300 g) the supernatant discarded, and resuspended in a reduced calcium content MNase buffer (Tris-HCl pH 7.5 10 mM; CaCl2 1 mM). Cells were split into 3 separate digests to allow titration of MNase (NEB M0247) concentration, which typically ranged from 5 to 20 Kunitz units per reaction. This reaction was incubated for 1 hour at 37 °C in an Eppendorf Thermomixer at 500 rpm and was quenched by adding ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA) (Sigma E3889) to a final concentration of 5 mM. Chromatin was decrosslinked (proteinase K, 65 °C, >2 hours) and DNA was purified using the Qiagen DNeasy blood and tissue kit (69506). Digestion and ligation efficiencies were assessed using the Agilent Tapestation (D1000 reagents). MNase digested samples were sequenced by ligating sequencing adaptors using the NEB Ultra II reagents (NEB, 7645S) without a size-selection step. The samples were sequenced using Illumina-based sequencing with 300-bp reads (150-bp paired-end reads).