GSM5005295: anti-PPAR-γ Cut-and-run Seq; Mus musculus; OTHER
Murine TCRβ+CD4+CD25hiYFP+ adipose Treg cells were sorted from 4-month old male Foxp3Cre mice. Treg cells were washed and bound to Concanavalin A-coated magnetic beads, then permeabilized with Wash Buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM spermidine and protease inhibitor) containing 0.05% digitonin (Dig Wash), and incubated with specific primary antibody overnight at 4℃. Cell-bead slurry was washed twice with Dig Wash, incubated with Protein A-MNase (pA-MN) for 1 hour at 4℃, then washed twice more with Dig Wash. Slurry was then placed on an ice-cold block and incubated with Dig Wash containing 2 mM CaCl2 to activate pA-MN digestion. After an antibody-specific incubation period, one volume of 2× Stop Buffer (340 mM NaCl, 20 mM EDTA, 4 mM EGTA, 0.05% Digitonin, 0.05 mg/mL glycogen, 5 μg/mL RNase A, 2 pg/mL heterologous spike-in DNA) was added to stop the reaction, and fragments were released by 30-minute incubation at 37℃. Samples were centrifuged 5 minutes at 16000xg, and supernatant was recovered. DNA was extracted via the DNA Purification Buffers and Spin Columns (CUT&RUN, Cell Signaling Technology, Cat#14209). Resulting DNA was subjected to sequencing. anti-HIF-1α (Novus, Cat#NB100-105) and anti-PPAR-γ (Abcam, Cat#ab41928) antibodies were used. Finally, DNA libraries were amplified and further sequenced on the Illumina Hiseq x10 platform.