NextSeq 500 paired end sequencing; Eight in situ Hi-C experiments of mouse OG2 stem cells for 7 different starting material and that were digested by either HindIII (2 samples) or MboI (6 samples) restriction enzymes
Eight in situ Hi-C experiments of mouse OG2 stem cells for 7 different starting material and that were digested by either HindIII (2 samples) or MboI (6 samples) restriction enzymes
1.We followed the general protocol for in situ Hi-C as described in (Rao et al., 2014) which we adapted for use on low cell numbers. Mainly, differences were related to adjustments on the volume of the reactions, a shortening of the digestion step, a removal of biotin from the un-ligated fragments, and an alternative strategy for size-selection during library preparation.  mESC OB2 cells were cultured as described in (Osunkoya et al., 1968)), FACS-sorted, selected for positive GFP expression and collected on PBS. Cells were then pelleted (300 g, 40C for 10 min) and resuspended in 1 ml PBS. 2. mESC OB2 cells were cultured as described in (Osunkoya et al., 1968)), FACS-sorted, selected for positive GFP expression and collected on PBS. Cells were then pelleted (300 g, 40C for 10 min) and resuspended in 1 ml PBS. 3.Cross-linking. In a fume hood, cells were cross-linked on a 1% final concentration (v/v) of 37% Formaldehyde and vigorously shaken prior to 10 min room temperature incubation with gentle rotation (20 rpm). The reaction was quenched for 5 min at room temperature and with gentle rotation (20 rpm) by adding 2.5M Glycine solution to a final concentration of 0.2M. Cells were twice pelleted (300 g, 4C for 5 min) and resuspended on 1 ml of cold 1X PBS. After these washes, cells were finally resuspended in 50 ml, quantified on a Neubauer's chamber and aliquoted at different cells concentrations (5M, 1M, 100k, 50k, 25k, 10k and 1k). 5. DNA shearing. DNA was precipitated by adding 630 μL Phenol-Chloroform-Isoamyl alcohol (25:24:1) and samples were spun (5 min at 17,000 g). The supernatant was then mixed to 63 μL of 3M sodium acetate pH5.2, 2 μL GlycoBlue (Life Technologies) and 1008 μL pure ethanol, followed by 15 min incubation at -80C. Samples were then spun at 17,000 g at least for 30 min. The supernatant was discarded and the pellet was washed with cold 70% ethanol. All ethanol traces were removed, the pellet was air-dried for no longer than 5 min and then was dissolved in 30 μL (5M to 100k samples) or 15 μL (50k to 1k samples) of Tris pH 8.0 (Applichem) and incubated for 5 min at 370C without rotation. RNA was digested by adding 1 μL RNAse to each sample and the mix was incubated 15 min at 370C. Biotin from unligated fragments was removed by adding 10 μL (5M to 100k samples) or 5 μL (50k to 1k samples) of 10X NEB2 buffer (New England Biolabs), 1mM of a dNTPs mix (2.5 μL for the 5M to 100k samples or 1.25 μL for the 50k to 1k samples), 20mg/ml BSA (0.5 μL for 5M to 100k samples or 0.25 μL for 50k to 1k samples), nuclease-free water (up to 100 μL or 50 μL, respectively) and 3U/μL T4 DNA polymerase (5 μL or 3 μL, respectively; New England Biolabs). Samples were mixed by gentle pipetting up and down and incubated at 200C for 4h with no rotation. Samples were brought to a 120 μL volume with nuclease-free water and DNA was then sheared using a Covaris S220 instrument (2 cycles, each 50 sec long; 10% duty; 4 intensity; 200 cycles/burst). Biotin Pull-down. Dynabeads MyOne Streptavidin C1 beads (Thermo Fisher Scientific) were used to pull down biotinylated fragments according to manufacturers guidelines (https://tools.thermofisher.com/content/sfs/manuals/dynabeads_myone_savC1_man.pdf). Briefly, 120 μL of the sheared Hi-C sample was mixed with an equal amount of the prepared magnetic beads and incubated for 15 min with rotation to allow DNA binding to the beads. The beads were washed twice with 1x B&W buffer + 0.1% Triton X-100 by incubating the samples at 550C with shaking (1000 rpm) for 2 min, followed by a wash with 10 mM Tris-Cl pH 8.0 and finally resuspended in 50 μL of the last wash solution. 4. Lysis. Cells were pelleted and gently resuspended in 500 μL (5M and 1M samples), 250 μL (100k, 50K, 25k and 10k samples), or 125 μL (1k sample) of ice-cold in situ Hi-C lysis buffer (10 mM Tris-Cl pH 8.0, 10 mM NaCl, 0.2% IGEPAL CA-630, cOmplete Ultra protease inhibitors). Following 15 min incubation on ice, cells were spun down (1000 g, 4C for 5 min) and pellet was resuspended in 250 μL (5M and 1M samples), 125 μL (100k, 50K, 25k and 10k samples) or 62.5 μL (1k sample) of ice-cold in situ Hi-C lysis buffer. Lysed cells were then flash-frozen in liquid Nitrogen and stored at -80C.Restriction Enzyme digestion. Frozen aliquots at different cell concentrations were placed on ice to thaw, spun (13,000 g for 5 min at 40C) and resuspended in 500 μL (5M and 1M samples), 250 μL (100k sample), 125 μL (50K, 25k and 10k samples), or 62.5 μL (1k sample) lysis buffer. Following yet another spun (13,000 g for 5 min at 40C), cells were gently resuspended in 500 μL (5M and 1M samples), 250 μL (100k sample), 125 μL (50K, 25k and 10k samples) or 62.5 μL (1k sample) ice-cold 10x NEB2 buffer. Nuclei were permeabilised by resuspending them in 50 μL (5M, 1M and 100k samples), 25 μL (50K, 25k, 10k) or 12.5 μL (1k sample) of 0.4% SDS and incubating for 10 min at 65C without agitation. The SDS was then quenched by adding 25 μL (5M, 1M and 100k samples) or 12.5 μL (50K, 25k, 10k and 1k samples) of 10% Triton X-100 and 145 μL or 72.5 μL of nuclease-free water, respectively, and incubating at 370C for 45 min with rotation (650 rpm). Chromatin was digested by adding either a) 500U of HindIII-HF (5M, 1M and 100k) in 25 μL of 10x NEB2 buffer, or b) 100U (5M, 1M and 100k samples), 50U (50k, 25k and 10k samples) or 25U (1k sample) of MboI in 25 μL (5M, 1M and 100k samples), 35 μL (50k, 25k and 10k samples) or 42.5 μL (1k sample) of 10x NEB2.1 buffer, respectively (New England Biolabs). All the digestions were performed at 370C with gentle rotation for a period of 90 min by adding the restriction enzyme in two instalments. After the digestion, MboI samples were heat-inactivated for 20 min at 62C.Marking of DNA Ends. The overhangs generated by the restriction enzyme cuts were filled-in by adding a mix of 0.4 mM biotin-14-dA/CTP (18.75 μL for the 5M to 100k samples or 10 μL for the 50k to 1k samples; Life Technologies), 10mM dA/CTP, 10mM dGTP and 10mM dTTP (0.75 μL of each dinucleotide for the 5M to 100k samples or 0.5 μL for the 50k to 1k samples), and 5U/μL DNA polymerase I Klenow (5M to 100k samples or 4 μL for the 50k to 1k samples; New England Biolabs), followed by a 90 min incubation at 370C with gentle rotation.Proximity ligation. The DNA fragments were then ligated by adding a master mix containing nuclease-free water (657 μL for the 5M to 100k samples or 328.5 μL for the 50k to 1k samples), 10X T4 DNA ligase buffer (120 μL for the 5M to 100k samples or 60 μL for the 50k to 1k samples), 10% Triton X-100 (100 μL for the 5M to 100k samples or 50 μL for the 50k to 1k samples), 20mg/ml BSA (12 μL for the 5M to 100k samples or 6 μL for the 50k to 1k samples) and 5 Weiss U/μL T4 DNA ligase (5 μL for the 5M to 100k samples or 3.5 μL for the 50k to 1k samples in two instalments; Thermo Fisher Scientific). Samples were mixed by inversion and incubated at 20C with gentle rotation for 4 h.Crosslink reversal. The crosslink was reverted by spinning down the nuclei (2500 g for 5 min at room temperature) and resuspending them in 500 μL (5M to 100k samples) or 250 μL (50k to 1k samples) extraction buffer. To digest the proteins 20 μL (5M to 100k samples) or 10 μL (50k to 1k samples) of 20mg/ml proteinase K (Applichem) was added to the mix, followed by 30 min incubation at 55C with rotation (1000 rpm). 130 μL (5M to 100k samples) or 65 μL (50k to 1k) of 5M sodium chloride was added followed to the mix and was incubated overnight at 65C with shaking (1000rpm).
Postdoctoral Fellow
E-MTAB-5875:100k-HindIII_p
Experimental Factor: starting material
100,000 cells
Experimental Factor: restriction digest
HindIII