GSM4200152
GSM4200152: Input, Hmga2-/-; Mus musculus; MNase-Seq
The nuclei were resuspended in Low Salt Buffer (10 mM Tris-HCl pH 7.4, 0.2 mM MgCl2 supplemented with protease and phosphatase inhibitors) including 1% Triton-X100. After 15 min incubation on ice, cells were spun down and the chromatin was resuspended in MNase digestion buffer with 1,250 Units MNase (NEB Biolabs). Chromatin-MNase mix was incubated at 37°C for 30 min. MNase reaction was stopped by adding 50 mM EDTA. Samples were sonicated for 30 sec on / 30 sec off using the Bioruptor with high amplitude. Chromatin was spun down at 14,000 rpm at 4°C for 10 min and the supernatant was used for ultracentrifugation. Sucrose gradients (5%-40%) were prepared in 1,800 μl low salt buffer (10 mM NaCl, 10 mM Tris-HCl pH7.4, 0.2 mM EDTA, 0.2 mM DTT, 20 mM NaF, 20 mM Na3VO4, 40 μg/ml phenylmethylsulfonyl fluoride and protease inhibitor) in polyallomer centrifuge tube (Beckman). Fragmented native chromatin was loaded on top of the 9 ml 5%-40% sucrose gradients and centrifuged for 16 h and 30 min at 37,500 rpm in an SW50.1 ultracentrifuge rotor (Beckman Coulter). Following centrifugation, 11 fractions (1000 μl each) were collected manually from the bottom of the tubes. From fractions 3 and 4, 200 µl  were treated with RNase A and purified using Phenol:Chloroform. Purified DNA was quantified by Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific). 10 ng DNA was used as input for TruSeq ChIP Library Preparation Kit (Illumina) with following modifications. Instead of gel-based size selection before final PCR step, libraries were size selected by SPRI-bead based approach after final PCR with 18 cycles. In detail, samples were first cleaned up by 1x bead:DNA ratio to eliminate residuals from PCR reaction, followed by 2-sided-bead cleanup step with initially 0.6x bead:DNA ratio to exclude larger fragments. Supernatant was transferred to new tube and incubated with additional beads in 0.2x bead:DNA ratio for eliminating smaller fragments, like adapter and primer dimers. Bound DNA samples were washed with 80% ethanol, dried and resupended in TE buffer. Library integrity was verified with LabChip Gx Touch 24 (Perkin Elmer).
GEO
GSM4200152
GEO Accession
GSM4200152