GSM8775391: Mesothelium_H3K27ac_100k_Rep2; Mus musculus; OTHER
For CUT&RUN, Timed Myrf Fl/Fl mating's were set up and pregnant E16.5 females were euthanized at E16.5. Lungs were pooled and and dissociated into single cells. After dissociation, cells were resuspended at a max staining volume of 50 million cells/mL. Cells were blocked with BD FC block (1:200, BD Biosciences) for 20 minutes on ice. Cells were stained with the following antibodies: APC/Cyanine7 anti-mouse CD326 (1:500, Biolegend), APC anti-mouse Podoplanin (1:500, Biolegend), FITC anti-mouse CD31 (1:50, Biolegend), Brilliant Violet 510™ anti-mouse CD45 (1:100) for 25 minutes on ice. Two washes with DPBS+ 0.2% FBS were performed, and prior to sorting samples were resuspended in DPBS + 0.2% FBS and DAPI(1:16,000). Samples were sorted using the BD FACSAria II (BD Biosciences). Mesothelial Cells were sorted as PDPN+EPCAM-CD45-CD31-DAPI- and Epithelial Cells were sorted as PDPN+EPCAM+CD45-CD31-DAPI-. Approximately 60-100k Mesothelial cells were collected and approximately 100-500k Epithelial cells were collected. CUT&RUN was performed using the CUT&RUN Assay Kit (CST) according to manufacturer's instructions. Live sorted 60-100k mesothelial and epithelial cells were used to profile H3K27ac and 500K epithelial cells were used to profile the IgG negative control. DNA library prep was performed using the KAPA HyperPrep Kit (Roche) and sequenced on the NovaSeq 6000 with 150 bp Paired End Reads