GSM8150515: MicroC_Bcells_WT_rep1; Mus musculus; OTHER
cells, isolated from WT and Het mice, were activated for 72hours by incubation with LPS and IL4 (20 µg/ml LPS and 20 ng/ml IL-4 final concentration) prior to experiment. Next, cells were collected by trypsinization, washed in PBS and counted. For crosslinking, 300 mM DSG (ThermoScientific) solution in DMSO (100x) was prepared freshly and diluted to 1x in room temperature PBS. Cellular pellet was resuspended in DSG solution and incubated for 35 minutes on rotating wheel at room temperature. Next 16% methanol-free formaldehyde (Thermo Scientific) was added to a mixture to a final concentration of 1% and incubated for next 10 minutes on rotating wheel at room temperature. To quench the crosslinking reaction, Tris pH 7.5 was added to a final concentration of 0.375 M and incubated on rotating wheel for 5 minutes at room temperature. Next, cells were centrifuged at 4oC, washed once with ice-cold PBS and re-counted. Cells were divided into 5 mln cells portions, pelleted, snap frozen and stored at -80°C until needed. MicroC was performed according to (Goel et al, 2023) with minor modifications. 10 million crosslinked activated B cells from WT and Het mice were resuspended in MB#1 buffer (10 mM Tris pH 7.5, 50 mM NaCl, 5 mM NgCl2, 1mM CaCl2, 0.1% Igepal, EDTA-free protease inhibitor cocktail (Roche)) and incubated on ice for 20 minutes to isolate nuclei. After one wash with MB#1 buffer, nuclei were resuspended in MB#1 buffer containing MNAse and incubated for 20 minutes in 37oC with 1000 rpm rotation in thermoblock to digest chromatin. Reaction was stopped by addition of 4mM EGTA and 10 minutes incubation at 65oC. Next, nuclear pellet was washed twice in MB#2 buffer (10 mM Tris pH 7.5, 50 mM NaCl, 10 mM MgCl2, BSA), resuspended in 100 ul Repair End Master Mix (1x NEB 2.1, 2mM ATP, 5mM DTT, 50 U PNK), incubated at 37oC for 15 mins with shaking 1000 rpm. Next, 50 U Klenow was added and incubated for next 15 mins at 37oC with shaking 1000 rpm. To biotinylate, Biotin Master Mix (66 nM Biotin-dATP, 66 nM Biotin-dCTP, 66 nM dTTP, 66 nM dGTP, T4 DNA ligase buffer, 33 ug BSA) was added and incubated 45 mins 25oC with shaking 1000 rpm. To quench the reaction EDTA was added to final concentration of 30 mM, incubated at 65oC for 20 minutes and the chromatin pellet was washed once with MB#3 buffer (50 mM Tris pH 7.5, 10 mM MgCl2, 50 ug BSA). Next, the chromatin pellet was resuspended in Ligation Master Mix (T4 buffer, 100 ug BSA, 10 000 U T4 DNA ligase), incubated for 2.5 hours at 25oC and centrifuged. The chromatin pellet was resuspended in Exonuclease Master Mix (NEBuffer 1, 1000 U Exonuclease III), incubated for 15 mins at 37oC and centrifuged. Next, the samples were incubated overnight at 65oC in Reverse crosslinking solution (1% SDS, 200 mM NaCl, 520 ug Proteinase K, 26 ug RNAseA), DNA was purified on 1.0x AmPure beads to enrich for dinucleosome fraction. Prior to library preparation 10-15ug biotinylated chromatin was pull-down using Dynabeads MyOne Streptavidin T1 for 30 mins incubation at room temperature, washed twice with 1xTWB (5 mM Tris-HCl pH 7.5, 0.5 mM EDTA pH 8.0, 0.1% Tween-20) and once with 10 mM Tris pH 7.5. Library was prepared using NEBNext® Ultra™ II DNA Library Prep Kit for Illumina® according to manufacturer protocol.