title: GSM9491676: Micro-C, CBX2_WT_rep3; Mus musculus; Hi-C
library_construction_protocol: Micro-C experiments were performed using a Dovetail® Micro-C Kit (Cantata Bio). 1.0 × 10⁶ cells were frozen at -80°C for at least 30 minutes and then thawed at room temperature for 5 minutes. The thawed pellet was resuspended in 200 µL of the freshly prepared DSG solution and nutated for 10 minutes at room temperature. Following nutation, 5.4 µL of 37% formaldehyde was added to a final concentration of 1%, and the mixture was nutated for an additional 10 minutes. After washing with 1× Wash buffer, the cells were resuspended in 50 µL of a MNase digest master mix (50 µL 1× Nuclease Digest Buffer + 0.5 µL MNase Enzyme Mix). The tube was then incubated in an agitating thermal mixer at 37°C at 1250 RPM for 18 minutes for in situ MNase digestion. After digestion, the reaction was stopped by adding 5 μL of 0.5 M EGTA, and QC was performed to confirm a 50-90% mono-nucleosome digestion profile. 3 μL of 20% SDS was added to the digested samples. The mixture was then incubated at 22°C for 5 minutes in the thermal mixer at 1250 RPM. Up to 1500 ng of lysate per sample was mixed with Chromatin Capture Beads. Once mixed, samples were incubated at room temperature for 10 minutes. The beads were collected on the magnetic rack and then washed 3 times with 1× Wash buffer. 53 μL of an End Polishing Master Mix (50 μL End Polishing Buffer + 3 μL End Polishing Enzyme Mix) was added to the washed sample. After the end polishing, 50 µl Bridge Ligation Mix was added to the beads, followed by 1 µl Bridge Ligase. Once thoroughly mixed, the reactions were incubated at 22°C for 30 minutes in an agitating thermal mixer set at 1,250 rpm. Following the bridge ligation, 50 μL Intra-Aggregate Ligation Mix was added to each sample. The samples were mixed and incubated at 22°C for 4 hours in an agitating thermal mixer at 1,250 rpm. After intra-aggregate ligation, samples were resuspended with 51.5 µL of Crosslink Reversal Solution (55 µL Crosslink Reversal Buffer + 1.7 µL Proteinase K) and incubated at 55°C for 10 minutes, then the temperature was increased to 65°C for overnight. DNAs were purified using SPRIselect beads and eluted with 95 μL TE Buffer pH 8.0. The eluted DNAs were quantified using a Qubit fluorometer and dsDNA HS kit. For Micro-C 150 ng of purified DNA was used to prepare a library for each replicate using the NEBNext Ultra II kit. 25 μL of Streptavidin beads was washed and mixed with 95 μL of purified and adaptor-ligated DNA. The beads were then washed with LWB, NWB, and 1× Wash buffer. 45 µL Index PCR Master Mix (25 µL Hot Start PCR Ready Mix + 20 µL water per sample) was added to the beads, followed by 5 µL of a unique UDI Primer Pair. The PCR reactions were performed with the following settings: Enzyme activation at 98°C for 3 minutes, followed by 12 cycles of denaturation at 98°C for 20 seconds, annealing at 65°C for 30 seconds, extension at 72°C for 30 seconds, and a final extension at 72°C for 1 minute. After amplification, 47 μL of the supernatant was collected, and 53 µL of TE Buffer (pH 8.0) was added to the solution, followed by 50 μL of SPRIselect beads. 145 µl of the supernatant was collected and mixed with 30 μL of SPRIselect beads. After washing, the beads were air-dried, and the DNA fragments were eluted with 30 μL TE Buffer (pH 8.0). The final elution was quantified with a Qubit fluorometer and a dsDNA HS kit, and the fragments were analyzed on a Tapestation to confirm a size distribution of 350-1,000 bp. NSG was performed to obtain a total of 300 million to 800 million reads between the replicates for each condition