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. Plus two peripheral blood mononuclear cells (PBMCs) samples obtained either from a blood extraction of a healthy donor or from a lymph node biopsy from a DLBCL patient.
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. Plus two peripheral blood mononuclear cells (PBMCs) samples obtained either from a blood extraction of a healthy donor or from a lymph node biopsy from a DLBCL patient.
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. For the human samples, PBMCs were crosslinked and MACS-sorted to isolate CD20+ B-cells to get the Hi-C libraries. Samples from a healthy donor and cancer patient processing. Patient and Control samples processing. Peripheral blood mononuclear cells (PBMCs) were obtained either from a blood extraction from a healthy donor or from a lymph node biopsy from a DLBCL patient. The patient sample came from the Department of Clinical Pathology at the Robert-Bosch-Hospital in Stuttgart (Germany) and its informed consent for retrospective analysis was approved by the ethics committee of the Medical Faculty, Eberhard-Karls-University and University Hospital Tübingen (reference no. 159/2011BO2). PBMCs from the control came from a donor from the Department of Medicine A, Hematology, Oncology and Pneumology, University Hospital Muenster in Muenster (Germany). Control PBMCs were isolated from the in-between layer by density gradient centrifugation with Biocoll (Biochrom AG, Germany) and were then frozen at -80°C for preservation. The patient sample came from a biopsy of a lymph node. Briefly, the biopsy was immediately cut into pieces, homogenized and re-suspended generating a cell suspension that was then frozen and kept at -80°C, as previously described at Staiger et al., 2017. Once the samples were thawed, cells were cross-linked in a 1% formaldehyde and quenched with 2.5M Glycine solution and proceed with the Low-C protocol as described for the rest of the samples. B-cells were then isolated by MACS-sorting (Yan et al., 2009) using a positive selection kit (Miltenyi Biotec, 130-091-104). Briefly, CD20+ cells were labelled using magnetic coated CD20 MicroBeads, the cell suspension was loaded onto a MACS LS column (Miltenyi Biotec, 130-041-306) and placed on a magnetic field generated by a MACS Separator. The CD20+ cells were retained into the column while the flow through (unlabelled cells) was eliminated. Then the column was removed from the MACS Separator, the magnetically retained CD20+ cells were then eluted and collected into a 15 ml Falcon tube.  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. Cells were then cross-linked in a 1% final concentration (v/v) of 37% Formaldehyde (VWR International GmbH) 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 with gentle rotation (20 rpm) by adding 2.5M Glycine solution (Applichem) to a final concentration of 0.2M. Cells were pelleted twice (300 g, 4°C 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 cell concentrations (5x106, 106, 105, 5x104, 2.5x104, 104 and 103 cells). 5. DNA shearing. DNA was precipitated by adding 630 μL (5M to 100k samples) or 315 μL  (50k to 1k samples) of the Phenol-Chloroform-Isoamyl alcohol mix (25:24:1; Sigma-Aldrich). Samples were spun (5 min at 17000 g, 4°C). The supernatant was then mixed to 63 μL (5M to 100k samples) or 31.5 μL (50k to 1k samples) of 3M sodium acetate pH5.2, 2 μL GlycoBlue (Life Technologies) and 1008 μL (5M to 100k samples) or 504 μL (50k to 1k samples) pure ethanol, followed by 15 min incubation at -80°C. Samples were then spun at 17000 g, 4°C for at least 30 min. The supernatant was then discarded and the pellet was washed twice with ice-cold 70% ethanol (800 μL for 5M to 100k samples or 400 μL for 50k to 1k samples). All ethanol traces were removed, the pellet was then air-dried for no longer than 5 min and 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 37°C without rotation. RNA was digested by adding 1 μL RNAse (Applichem) to each sample and the mix was incubated for 15 min at 37°C. 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 20°C 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). 4. Lysis. Cells were pelleted (13,000 g, 4°C for 5 min) 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 (Sigma-Aldrich), cOmplete Ultra protease inhibitors (Roche)). Following 15 min incubation on ice, cells were spun down (1000 g, 4°C for 5 min) and the 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 -80°C. Restriction enzyme digestion. Frozen aliquots for different cell concentrations were placed on ice to thaw, spun (13,000 g for 5 min at 4°C) 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 spin (13,000 g for 5 min at 4°C), 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 (New England Biolabs). Then, nuclei were spun once more for 5 min at 13,000 g at 4°C before being 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 65°C without agitation. The SDS (Applichem) 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 (Applichem) and 145 μL or 72.5 μL of nuclease-free water, respectively, and incubating at 37°C for 45 min with rotation (650 rpm). Chromatin digestion was performed as follows: i) for HindIII digested samples, by adding 500U of New England Biolabs HindIII-HF (5M and 100k samples) in 25 μL of 10x NEB2 buffer; and ii) for MboI (New England Biolabs) digested samples, by adding 100U (1M and 100k samples), 50U (50k, 25k and 10k samples), or 25U (1k sample) of MboI in 20 μL (5M, 1M and 100k samples), 35 μL (50k, 25k and 10k samples) or 42.5 μL (1k sample) of 10x NEB2.1 buffer (New England Biolabs), respectively. All digestions were performed at 37°C with gentle rotation for a period of 90 min by adding the restriction enzyme in two instalments, the second one after 45 min. After digestion, only MboI samples were heat-inactivated for 20 min at 62°C. Marking of DNA ends. The overhangs generated by the restriction enzyme cuts were filled-in by adding a mix of biotin-14-CTP (0.4 mM stock; 18.75 μL for the 5M to 100k samples or 10 μL for the 50k to 1k samples; Life Technologies), 10mM dATP (whichever was not supplied in biotinylated form), dGTP and dTTP (10 mM stocks; 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 37°C with gentle rotation (20 rpm). Proximity ligation. The resulting 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; Thermo Fisher Scientific), 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; New England Biolabs) 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 20°C with gentle rotation (20 rpm) for 4 h. Crosslink reversal. Nuclei were then spun (2500 g for 5 min at room temperature) and resuspended in 500 μL (5M to 100k samples) or 250 μL (50k to 1k samples) extraction buffer to revert the crosslinking. To digest the proteins 20 μL (5M to 100k samples) or 10 μL (50k to 1k samples) of 20mg/ml proteinase K (Applichem) were added followed by 30 min incubation at 55°C with shaking (1000 rpm). Subsequently, 130 μL (5M to 100k samples) or 65 μL (50k to 1k) of 5M sodium chloride was added to the samples, which were then incubated overnight at 65°C with shaking (1000 rpm).
Postdoctoral Fellow
E-MTAB-58751531847201:B-cells from healthy donor_p
Experimental Factor: organism
Homo sapiens
Experimental Factor: disease
normal
Experimental Factor: starting material
50,000 cells
Experimental Factor: restriction digest
MboI