GSM8160091: CETN_ingAT_Ucp1ERCre_H3K4me3_3; 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˚C 30 seconds, 72˚C final extension for 1 minute, hold at 4˚C. After 5 cycles the PCR was put on hold at 4˚C and 10% of the reaction was transferred into a 384 well plate and mixed with KAPA SYBR 2x (qPCR) and primers and qPCR was run for 20 cycles to determine the number of PCR cycles to be added38. Libraries were cleaned using SPRI beads using a 1.3 ratio, eluted in nuclease free water and pooled equimolecularily after library quantification using the KAPA library quantification kit (Roche Diagnostics, #079602).