title: GSM2476419: Diploid_8; Mus musculus; OTHER
library_construction_protocol: For harvesting ESCs from cultures on feeder cells, the feeder cells were depleted using Feeder Removal MicroBeads (Miltenyi Biotec), followed by fixation and permeabilisation of the enriched ESCs and staining for Oct-3/4-immunoreactivity as described below, and Oct-3/4-positive cells were collected by Fluorescence-activated cell sorting (FACS), were ready for subsequent Hi-C processing.    To fix the cells, 50 to 200 million ESCs were suspended in relevant medium and fixed for 10 min by adding formaldehyde at a final concentration of 2% at room temperature before quenching with 127 mM glycine for 5 min on ice. The cells were washed with PBS and permeabilised in 10 mM Tris-HCl (pH 8), 10 mM NaCl, 0.2% IGEPAL CA-630 with cOmplete EDTA-free protease inhibitor cocktail (Roche) for 30 min on ice with intermittent agitation, and spun to collect the nuclei pellet, which was ready for Hi-C processing.    To enrich for specific cell types before the Hi-C process, cells were blocked with PBS-FT (5% FBS, 0.1% Tween-20 in PBS) at room temperature for 1 hr, incubated with a primary antibody in PBS-FT as required (anti-Oct3/4, Santa-Cruz sc-5279, at 1:100 dilution; or anti-Geminin, Abcam 175799 at 1:400 dilution) for 1 hr on ice. Samples were then washed with PBS-FT, and incubated with the secondary antibodies (Alexa Fluor 647-anti-mouse IgG, Thermo Fisher A-31571, or Alexa Fluor 555-anti-rabbit IgG, Thermo Fisher A-21428) for 30 min on ice, washed with 2% FBS in PBS, stained with Hoechst 33342 at the final concentration of 15 µg/ml and subjected to FACS by Influx (BD Biosciences) to collect nuclei cells of interest. Hi-C processing  The cells (approximately half million to several million) were washed with 1.24 x NEBuffer 3 (New England Biolabs; 62 mM Tris-HCl [pH 7.9], 124 mM NaCl, 12.4 mM MgCl2, 1.24 mM DTT) and suspended in 400 µl of 1.24 x NEBuffer 3. Six µl of 20% SDS was added and incubated at 37˚C for 60 min with constant agitation, then 40 µl of 20% Triton X-100 was added and incubated at 37˚C for 60 min with constant agitation. Next, 50 µl of 25 U/µl Mbo I (New England Biolabs) was added and incubated at 37˚C overnight with constant agitation. To label the digested DNA ends, 1.56 µl of 10 mM dCTP, 1.56 µl of 10 mM dGTP, 1.56 µl of 10 mM dTTP, 39 µl of 0.4 mM biotin-14-dATP (Thermo Fisher) and 10.4 µl of 5 U/µl DNA polymerase I, large (Klenow) fragment (New England Biolabs) were added and incubated at 37˚C for 45 min with occasional mixing. The sample was then spun and supernatant partially removed leaving 50 µl with cells, followed by addition of 100 µl of 10x T4 DNA ligase reaction buffer (New England Biolabs), 10 µl of 100x BSA (New England Biolabs), water and 10 µl of 1 U/µl T4 DNA ligase (Thermo Fisher) were added to make the total volume 1 ml, and incubated at 16˚C overnight. Then the nuclei were passed through a 30 µm cell strainer and single nuclei were sorted into individual empty wells in 96 well plates using an Influx cell sorter. The plates were sealed and stored at -80˚C until further processing.    Single-cell Hi-C library preparation  To prepare single-cell Hi-C libraries from single nuclei in 96 well plate, 5 µl of PBS was added to each well, the plate was sealed and crosslinks reversed by incubating at 65˚C overnight. Hi-C concatemer DNA was fragmented and linked with sequencing adapters using the Nextera XT DNA Library Preparation Kit (Illumina), by adding 10 µl of Tagment DNA Buffer and 5 µl of Amplicon Tagment Mix, incubating at 55˚C for 5 min, then cooling down to 10˚C, followed by addition 5 µl of Neutralize Tagment Buffer and incubation for 5 min at room temperature. Hi-C ligation junctions were then captured by Dynabeads M-280 streptavidin beads (Thermo Fisher; 20 µl of original suspension per single-cell sample). Beads were prepared by washing with 1 x BW buffer (5 mM Tris-Cl pH 7.5, 0.5 mM EDTA, 1 M NaCl), resuspended in 4 x BW buffer (20 mM Tris-Cl pH 7.5, 2 mM EDTA, 4 M NaCl; 8 µl per sample), and then mixed with the 25 µl sample and incubated at room temperature overnight with gentle agitation. Using a Bravo automated liquid handling system (Agilent Technologies), the beads were then washed four times with 200 µl of 1 x BW buffer, twice with 200 µl of 10 mM Tris-Cl pH 7.5 at room temperature, and resuspended in 25 µl of 10 mM Tris-Cl pH 7.5. Single-cell Hi-C libraries were amplified from the beads by adding 15 µl of Nextera PCR Master Mix, 5 µl of Index 1 primer of choice and 5 µl of Index 2 primer of choice. Samples were then incubated at 72˚C for 3 min, 95˚C for 30 sec followed by the thermal cycling at 95˚C for 10 sec, 55˚C for 30 sec and 72˚C for 30 sec for 12 or 18 cycles, then incubated at 72˚C for 5 min. The supernatant was separated from the beads, and the 96 supernatants from a 96 well plate that had 12 cycles of amplification were combined together whereas the supernatants from 18 cycles of amplification were processed uncombined. The combined or uncombined supernatant was purified with AMPure XP beads (Beckman Coulter; 0.6 times volume of the supernatant) according to manufacturer's instructions and eluted with 10 mM Tris-Cl pH 8.5 (100 µl when 96 samples were combined; 30 µl when sample was uncombined). The eluate was purified once more with AMPure XP beads (equal volume to the previous eluate) and eluted with 11 µl of 10 mM Tris-Cl pH 8.5.
attributes.tag: GEO Accession
attributes.value: GSM2476419