title: GSM4481174: Embryo Hi-C data 10; Mus musculus; Hi-C
library_construction_protocol: Pregnant C57BL/6 mice were sacrificed at day 9.5 post-coitum and embryos were dissected under a microscope. The yolk sack was mechanically removed from each embryo, leaving the embryo proper only, and the embryo morphology was validated to match that of a wildtype embryo of the appropriate age. To create single-cells suspension, each embryo was moved to a 1.5ml tube containing 200µl of trypsin-EDTA (0.05% trypsin, 0.02% EDTA) and incubated at 37ºC for 5 minutes. 800µl of cold MEF medium was then added to each tube to inactivate the trypsin. Cell suspensions of all embryos were combined and MEF medium at room temperature was added to a final volume of 21ml. 3ml of 16% formaldehyde were added (2% formaldehyde final concentration) and the mixture was incubated for 10 minutes at room temperature, followed by quenching with 127mM glycine for 5 minutes on ice and washing with cold PBS + 0.001% BSA. Cells were then permeabilized in 10 mM Tris-Cl pH 8, 10 mM NaCl, 0.2% IGEPAL CA-630 and cOmplete EDTA-free protease inhibitor cocktail (Roche) for 30 min on ice with intermittent agitation, and spun to collect a nuclei pellet.  The nuclei were washed with 1.24x NEBuffer 3 (New England Biolabs) and suspended in 400µl of that buffer. 6µl of 20% SDS and then 40µl of 20% Triton X-100 were added to the suspension, with an incubation of 60 minutes at 37ºC with constant agitation following the addition of each of these detergents. Next 50µl of 25U/µl MboI (New England Biolabs) was added and the suspension incubated at 37ºC overnight with constant agitation.  To label the digested DNA ends, dCTP, dGTP, dTTP and biotin-14-dATP (Thermo fisher) were added to the suspension (final concentration of 28.36µM per nucleoside triphosphate) along with DNA polymerase I, large (Klenow) fragment (New England Biolabs, final concentration 0.095U/µl) and the sample incubated at 37ºC for 60 minutes with occasional mixing. The sample was then spun and the supernatant partially removed, leaving a volume of 50µl, followed by the addition of 100µl 10x T4 DNA ligase reaction buffer (New England Biolabs), 10µl 100x BSA (New England Biolabs), 10µl of 1U/µl T4 DNA ligase (Thermo Fisher) and water to a final volume of 1 ml, and incubated at 16ºC overnight. Finally, the nuclei were filtered through a 30µm cell strainer and single nuclei were sorted into individual empty wells in 384 well plates using an BD Influx cell sorter. The plates were sealed and stored at -80ºC until further processing.  To prepare single-cell Hi-C libraries from single nuclei in plate wells, 2.5µl of PBS was added to each well and the plate was sealed and incubated at 65ºC overnight. DNA was then tagmented using the Nextera XT kit (Illumina) by adding 5µl of TD and 2.5µl of ATM per well and incubating at 55ºC for 5 minutes, followed by cooling to 10ºC and adding of 2.5µl of NT per well. Hi-C ligation junctions were then captured by Dynabeads M-280 streptavidin beads (Thermo Fisher; 10µl of original suspension per well). Beads were prepared by washing with 1x BW buffer (5mM Tris-Cl pH 7.5, 0.5 mM EDTA, 1M NaCl), resuspended in 4x BW buffer (20mM Tris-Cl pH 7.5, 2mM EDTA, 4M NaCl; 4µl per sample), and then mixed with the 12.5µl per-well sample and incubated at room temperature overnight with gentle agitation. The beads were then washed four times with 40µl of 1x BW buffer, washed twice with 40µl of 10mM Tris-Cl pH 7.5 and resuspended in 12.5µl of 10mM Tris-Cl pH 7.5. Single-cell Hi-C libraries were amplified from the beads by adding 7.5µl of Nextera PCR Master Mix, 2.5µl of Index 1 primer and 2.5µl of Index 2 primer (a different combination of index 1 and index 2 per well) followed by 12 PCR cycles. The beads were then magnetically removed and the supernatant from all 384 wells combined. The combined supernatant was purified using AMPure XP beads (Beckman Coulter; 0.6 times volume of the supernatant) according to the manufacturer's instructions and resuspended in 100µl of 10mM Tris-Cl pH 7.5. Finally, the sample was purified again using AMPure XP beads (1.0 times volume of supernatant) and resuspended in 11µl of 10mM Tris-Cl pH 7.5.