GSM8160090: CETN_ingAT_Ucp1ERCre_H3K4me3_2; Mus musculus; OTHER
Nuclei were isolated from snap frozen adipose tissue depots in ice cold Nuclei Extraction Buffer (Miltenyi, #130-128-024) supplemented with 10 mM sodium butyrate and 1x cOmpleteTM EDTA-free Protease Inhibitor (Roche, #5056489001) using the gentleMACSTM Octo Dissociator (Miltenyi, #130-096-427) using C-tubes (Miltenyi, #130-093-237). All centrifugation steps were carried out in a to 4˚C cooled swinging bucket centrifuge with settings at 500 xg for 5 minutes. Nuclei were subsequently filtered through a 50µm cell strainer (Sysmex, #04-0042-2317) and washed two times in PBS-BSA (1% w/v) containing protease inhibitor and sodium butyrate. Nuclei were centrifuged and subsequently bound to pre-blocked Dynabeads MyOne Streptavidin C1 beads (ThermoFisher, #65002) for 30 minutes at 4˚C followed by 3 washes with PBS-BSA (1% w/v) on a magnetic stand. CUT&Tag was performed as previously described with minor adjustments36,37. All buffers were supplemented with 1 x cOmplete EDTA-free Protease inhibitor and 10mM sodium butyrate. Briefly, nuclei bound to beads were aliquoted into 96-well LoBind plates (Eppendorf, #0030129547) and incubated with primary antibodies – anti-H3K4me3 (abcam, #ab8580), anti-H3K27me3 (Cell Signaling Technology, #C36B11), anti-H3K27ac (abcam, #ab4729), anti-H3K4me1 (abcam, #ab8895), anti-H3K9me3 (abcam, #ab8898) – overnight at 4˚C. All antibodies were diluted 1:100. With the plate on a magnet the primary antibody solution was removed, the beads were resuspended in secondary antibody solution (guinea pig anti-rabbit IgG (antibodies-online gmbh, #ABIN101961); 1:200 dilution) and incubated at room temperature for 30 minutes while rocking. pA-Tn5 was bound to antibodies, transposition performed at 37 ˚C and stopped using TAPS-Wash solution. Nuclei were lysed and pA-Tn5 decrosslinked using SDS-release solution. PCR was performed using Kapa HiFi plus dNTPs (Roche Diagnostics, #07958846001) with the following PCR settings: 72˚C 5 minutes, 98˚C 30 seconds, X cycles of 98˚C 10 seconds, 63˚