GSM6267405: ESC-2i_H3K18la_2; Mus musculus; OTHER
ESC were processed exactly as per the original CUT&Tag protocol for whole cells. All other samples were processed as per the CUT&Tag protocol for nuclei. Muscle samples were thawed and sliced in small pieces on ice. Subsequently, the pieces were transferred to an ice-cold dounce homogenizer (7 mL) and 3 mL of nucleus extraction buffer (1× prelysis buffer from the EpiQuick Total Histone Extraction Kit (EpiGentek)) was added before douncing, on ice, 10× with pestle A and 10× with pestle B. Nuclei from PIM was obtained through resuspension of centrifuged and washed cell pellets in the same nucleus extraction buffer. Nuclei from adipocytes were isolated using a Kimble 7 ml glass douncer using ice-cold nuclei isolation buffer (10 mM Tris-HCl pH 7.4, 3 mM MgCl2, 10 mM NaCl, 0.1 % Igepal-CA630, 1x protease inhibitor). To all samples: to stop the lysis reaction, of PBS+1%BSA was added in a 1:1 volume. After 5' centrifugation at 4˚C, 500 rpm, the supernatant was removed, the nuclei were resuspended in PBS+1%BSA and a sample was visually inspected for viability, purity and abundance of nuclei under the microscope. CUT&Tag was performed according to the published CUT&Tag sequencing protocol for nuclei.All buffers were supplemented with 5 mM sodium-butyrate and 1X complete protease inhibitor (Merck, 1187358000). Protein lo-bind tubes (Eppendorf, EP0030108116) were used to reduce sample loss.  Antibodies against H3K18la (PTM-Bio, PTM-1406), H3K4me3 (Abcam, ab8580), H3K27me3 (Cell Signaling Technology, C36B11), H3K27ac (Abcam, ab4729) and H3K9me3 (Abcam, ab8898) were used in this study. PCR was performed with 21 μL DNA + 2 μL Universal i5 primer (10 μM) + 2 μL uniquely barcoded i7 primers (10 μM) in a thin-wall 0.5 ml PCR tube, using a different barcode for each sample. *Nextera primers or indexed primers described by Buenrostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523:486 (2015).To this mixture,  25 μL NEBNext HiFi 2x PCR Master mix was added. The samples were mixed, quick spinned and placed in  a thermocycler with heated lid. The following program was used: Cycle 1: 72 °C for 5 min (gap filling) Cycle 2: 98 °C for 30 sec.Cycle 3: 98 °C for 10 sec.Cycle 4: 63 °C for 10 sec.Repeat Cycles 3-4 13 times 72°C for 1 min and hold at 8 °C.