GSM9709005: dTAG-NIPBL_dTAGV-1_titration_2nM RCMC; Mus musculus; OTHER
Micro-C library preparation was performed according to (Goel et al. 2023). Briefly, trypsinised cells were sequentially crosslinked with 3 mM disuccinimidyl glutarate (DSG, ThermoFisher, 20593) in PBS for 35 min at r.t. with gentle mixing, after which formaldehyde (ThermoFisher, 28926) was added to a final concentration of 1%. The double crosslinking reaction was incubated for further 10 min at r.t. with gentle mixing and quenched with Tris-HCl pH 7.5 at a final concentration of 0.375 M for 5 min. Aliquots of 5 x 106 cells were snap frozen in liquid nitrogen and stored at -80  C. Cell pellets were lysed into nuclei in complete MB1 (50 mM NaCl, 10 mM Tris-HCl pH 7.5, 5 mM MgCl2, 1 mM CaCl2, 0.2 % NP40, protease inhibitor cocktail) at 1 million cells/100   l for 20 min in ice. Nuclei were spun down at 1750 g for 5 min at 4  C and washed once with complete MB1. Chromatin was in situ fragmented at 37  C for 20 min with MNase (NEB, M0247S), titrated to yield mono- and dinucleosomes, in 100   l per 1 million cells of MB1. The appropriate amount of MNase was determined by a titration assay with the aim of digesting chromatin to 80-90% mononucleosomes/ 20-10% dinucleosomes (Goel et al. 2023). MNase digestion was stopped with EGTA at a final concentration of 4 mM. Fragment ends were repaired, biotin labelled and proximity ligated as described (Goel et al. 2023). After de-crosslinking and DNA extraction, dinucleosome DNA fragments were isolated and extracted form a 1% agarose gel. DNA ends were polished, blunted and ligated DNA was isolated by pulling down biotin-labelled fragments. Illumina library preparation was performed using NEBNext Ultra    II DNA Library Prep Kit for Illumina (NEB, E7103). Library PCR was performed using index primers (NEBNext Multiplex Oligos for Illumina, Dual Index Primers Set 1; NEB, E7600S) supplemented with 1   EvaGreen (Jena Bioscience, PCR-379). 1/5 of the volume was used to run a test qPCR to determine the appropriate number of cycles by which libraries are amplified to linear phase. Biotin-bait preparation using fragmented BACs covering the regions of interest was adapted from (Golov et al. 2020). 5   g of pooled equimolar amounts of the selected BACs (Suppl. table 5) were sonicated using MSE Soniprep 150 Plus at medium power for two 30 sec pulses on ice to shear DNA to a size of 150-700 bp. Equimolarity was calculated by qPCR using primers against the chloramphenicol resistance gene (Suppl. table 3). Forked adapters (Suppl. table 3) were annealed in NEBuffer 2 at 1X (NEB, B7002S) to a final concentration of 20   M in a thermal cycler set to heat at 98  C and gradually cool down to 4  C with a 1  C per 20 sec gradient. Shredded BACs were subjected to end-preparation, A-tailing and adapter ligation as described (Golov et al. 2020). BAC baits were PCR amplified using 5    biotinylated forward and reverse primers (Suppl. table 3) in the following reaction mix: 10   l 2X NEBNext Ultra    II Q5 Master Mix (NEB, M0544S), 1   l of each 10   M bio-primer, 2   l adapter-ligated BAC DNA, 1X EvaGreen. The number of amplification cycles was determined by the linear phase in a test qPCR. Around 3.5   g of biotinylated baits were obtained from 25 reactions. Enrichment of the regions of interest was adapted from (Golov et al. 2020). Equal amounts of all libraries corresponding to each sample, and amplified with different barcodes, were pooled and captured as a pool. One hybridization reaction was done per 1   g of library pool. If more than 4 samples were being captured, more than one hybridization reaction was performed. Hybridization and biotin-pulldown was done as described (Golov et al. 2020). Singly enriched 3C libraries were amplified by qPCR using p5/p7 primers (Suppl. table 3) as described above. PCR reactions were performed with the DNA still bound to the streptavidin beads. The number of amplification cycles was determined by the linear phase in a test qPCR. A second hybridization reaction, biotin pulldown and PCR amplification was performed as before. The pool was sequenced using Illumina Nextseq 2000 using a single P2 flow cell (paired-end 50-bp reads).