GSM4630482: pcHiC_Early_rep3; Mus musculus; Hi-C
Hippocampal tissue was extracted and flash frozen. After extraction, samples were immediately homogenized in 0.5 mL ice cold PBS with protease inhibitors (Pi) (11836170001, Roche). The suspension was fixed with 2% paraformaldehyde for 10 min, quenched with Glycine 2.5M for 5 min and washed twice with 5 mL of PBS. The suspension was centrifuged at 1200 g, 4°C, for 5 minutes and the pellet was resuspended in 5 mL NF-1 hypotonic buffer (0.5% Triton X-100/0.1M Sucrose/5mM MgCl2/1mM EDTA/10mM Tris-HCl, pH 8.0, Pi). Pelleted nuclei were resuspended in and incubated with NeuN and GFP primary antibodies for overnight in 4°C. Unbound antibodies were washed out twice and nuclei were filtered with 40 µm mesh filter for FACS sorting. Hi-C libraries were prepared from 10,000 cells /per group (N=3-4), as previously described (Nagano, T. et al. Nat. Protoc. 2015), with some modification. First restriction digestion was carried as described in the original protocol with 1,500 U of HindIII (R3104L, New England Biolabs, USA), as the 6-base cutter (instead of BglII in the original protocol). After Biotin labeling and overnight ligation, samples were spun down (4°C, 1,600 x g for 15 min) and the pellet was resuspended in 25 μl of PBS. The suspension was incubated in 65c with 3 μl of proteinase K for 12-16h. Purification of Hi-C DNA was carried as described in the original protocol with 100 μl of streptavidin-beads. For the second restriction enzyme we used the 4-base cutter Hpych4V (10 U /per sample, R0620S, New England Biolabs, USA). After A-tailing reaction (see original protocol for details), samples were amplified using PCR (17 cycles) and purified with AMPure XP beads. Hi-C libraries were eluted in 20 ul of 10 mM Tris-HCl (pH 8.5). Bait capture system design for Promoter Capture Hi-C – A list of 5000 mouse gene promoters was obtained from the previously published protocol in Schoenfelder, S. et al. Genome Res. 2015.  Selected probes were synthesized by Agilent Technologies according to the SureSelect target enrichment system (Agilent Technologies). To capture Hi-C ligation products containing promoter sequences, we used the previously published protocol (Schoenfelder, S. et al. Genome Res. 2015).
GEO Accession
GSM4630482