title: GSM8992039: mES-E14-WT-EnhancerBait_4; Mus musculus; OTHER
library_construction_protocol: 3C library manufacture was undertaken as described previously(Hamley et al., 2023). In brief, aliquots of 1 x 107 cells were fixed 10 ml media with Formaldehyde, 37% (vol/vol) (Sigma, 47608-250ML) at a final of concentration 2% for 10 minutes at room temperature. This reaction was quenched with 1 M glycine to a final concentration 130 mM and centrifuged (5 min, 300 g). The supernatant was removed, and the cell pellet washed with cold PBS and reconstituted in 1 ml PBS. Cells were permeabilised with Digitonin (Sigma D-141; final concentration of 0.005% wt/vol). Cells were snap frozen and stored at -80 °C. Subsequently, defrosted, permeabilised cells were centrifuged (5 min, 300 g) the supernatant discarded, and resuspended in a reduced calcium content MNase buffer (Tris-HCl pH 7.5 10 mM; CaCl2 1 mM). Cells were split into 3 separate digests to allow titration of MNase (NEB M0247) concentration, which typically ranged from 5 to 20 Kunitz units per reaction. This reaction was incubated for 1 hour at 37 °C in an Eppendorf Thermomixer at 500 rpm and was quenched by adding ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA) (Sigma E3889) to a final concentration of 5 mM. To evaluate digestion efficiency, 20% of the reaction was removed. The remaining reaction was centrifuged (5 min, 300 g) and the supernatant containing digestion buffer was discarded. The cell pellet was washed in 1 ml PBS and the cells were resuspended in DNA ligase buffer (Thermo Scientific; final concentrations Tris-HCl pH 7.5 40 mM, MgCl2 10 mM, DTT 10 mM, 5 mM ATP) supplemented with dNTPs (final concentration 400 µM of each of dATP, dCTP, dGTP and dTTP (Thermo Fisher R0191)) and EGTA 5 mM. The reaction mixture was then treated with T4 Polynucleotide Kinase PNK (NEB M0201L) and DNA Polymerase I Large (Klenow) Fragment (NEB M0210L) at final concentrations of 200 U/ml, and 100 U/ml respectively, T4 DNA ligase (Thermo Scientific, High Concentration Ligase (30 U/µl) EL0013) was added to a final concentration of 300 U/ml and the reaction was incubated at 20°C overnight using an Eppendorf Thermomixer at 500 rpm. Chromatin was decrosslinked (proteinase K, 65 °C, >2 hours) and DNA was purified using the Qiagen DNeasy blood and tissue kit (69506). Digestion and ligation efficiencies were assessed using the Agilent Tapestation (D1000 reagents). Sequencing library preparation. Oligonucleotide capture was performed as previously described(Hamley et al., 2023; Hua et al., 2021a). Initially, 7-10 µg of each 3C library was sonicated to an averagefragment size of 200 bp using a Covaris S220 Focussed Ultrasonicator (3 cycles of 60 seconds, duty cycle 10%, intensity 5, 200 cycles per burst). After sonication, the size of library was evaluated using an Agilent TapeStation (DNA 1000). The sample was then cleaned using Ampure XP beads (Beckman Coulter A63881). To maximise library complexity, three separate aliquots of 2 µg each of the sonicated 3C library were processed simultaneously for the addition of sequencing adaptors (NEB Ultra II (NEB 7645S)) following the standard protocol with the following alternations: 7.5 µl adapter was used in ligation reactions; all Ampure XP bead clean ups were performed at a ratio of 1 : 1.8 and  the final PCR was carried out in triplicate using the Herculase II PCR reagents (Agilent) to enhance DNA yield.