title: GSM4880323: CTL_Adipose_MicroC_Rep2; Mus musculus; OTHER
library_construction_protocol: Micro-C libraries with adipose tissue were prepared as previously described (Krietenstein et al., Molecular Cell, 2019). Briefly, for each replicate library, 2 million cells were crosslinked with 1% formaldehyde for 10 min at room temperature. Formaldehyde crosslinking was quenched with 0.25 M glycine for 5min at RT. After washing with 1xPBS, resuspended cells were further crosslinked with 3mM DSG in PBS for 40 min at RT. Crosslinking was quenched with 0.4M glycine for 5min at RT. Cells were resuspended in cold 1x PBS and incubated on ice for 20 min. After collecting cells by centrifugation, cells were washed with buffer MB#1 (10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 5 mM MgCl2, 1 mM CaCl2, 0.2% NP-40, 1x Roche cOmplete EDTA-free). Chromatin was fragmented with MNase for 10 min at 37°C and digestion was stopped by 5 mM M EGTA at 65°C for 10 min. The chromatin was resuspended in 1x NEBuffer 2.1  and dephosphorylated by addition of 5 μl rSAP at 37°C for 45 min. 5′ overhangs were generated with this pre-mix (50 mM NaCl, 10 mM Tris, 10mM MgCl2, 100 µg/ml BSA, 2 mM ATP, 3 mM DTT 8 μl Large Klenow Fragment and 2 μl T4 PNK) at 37°C for 15 min. The DNA overhangs were filled with biotinylated nucleotides by addition of 100 μl pre-mix (25 μl 0.4 mM Biotin-dATP, 25 μl 0.4 mM Biotin-dCTP, 2 μl 10 mM dGTP and 10 mM dTTP, 10 μl 10x T4 DNA Ligase Reaction Buffer, 0.5 μl 200x BSA, 38.5 μl H2O) and incubation at 25°C for 45 min. The reaction was stopped by addition of 12 μl 0.5 M EDTA at 65°C for 20 min. After proximity ligation and removing unligated ends, the DNA was first phenol/chloroform purified, second purified with DNA clean & Concentrator Kit, and then library was purified via 1.5% agarose gel electrophoresis. Dynabeads™ MyOne™ Streptavidin C1 beads were added to the sample and incubated at RT for 20 min. The beads were washed twice and resuspended in 50 ul TE buffer. Sequencing library was prepared with NEBNext Ultra II DNA Library Prep Kit for Illumina according to protocol, and PCR amplification, sample indexing, and DNA purification after PCR was performed.