GSM8306059: NPC cells,cKO,E15.5,K4me3_biol rep 2; Mus musculus; OTHER
E15.5 embryonic kidneys were dissected and cut into small pieces, trypsinized for 30 min at 37℃, and trypsinization was terminated by adding DMEM/F12 supplemented with 10% FBS. After centrifugation, cells were resuspended in DMEM/F12 supplemented with 2% FBS and filtered 2 times through 70µm cell-strainer cap (Biosharp, BS-70-XBS). The single cell suspension was transferred into a polypropylene tube (Falcon, 352063) and kept on ice until FACS. The GFP+ cells were isolated into DMEM/F12 supplemented with 10% FBS using Sony MA900 Cell Sorter. CUT&Tag-seq, For library preparation, the NovoNGS® CUT&Tag 3.0 High-Sensitivity Kit was purchased from Novoprotein, and libraries were generated according to the manufacturer's instructions. In brief, 1.0×104 cells were bound to ConA-coated beads and incubated with primary antibody against H3K4me3 (diluted 1:50 in primary antibody buffer) for 2 hours at room temperature. The beads were washed several times in wash buffer, incubated at room temperature for 1 hour using goat anti-rabbit IgG secondary antibody (diluted 1:50 in antibody buffer; catalog no. 31210, Invitrogen), again washed several times in wash buffer, then incubated with ChiTagTM pAG-Tn5 transposon (diluted 1:50 in ChiTag buffer) for 1 hour at room temperature. Tagmentation buffer containing MgCl2 was added, and the mixture was incubated at 37 ℃ for 1 hour. Tagmentation was stopped by adding sodium dodecyl sulfate and heating the mixture at 55 °C for 10 minutes. DNA fragments were purified using Tagment DNA Extraction Beads and PCR-amplified in reactions containing 33 μl extracted DNA, 2.5 μl primer i5 and 2.5 μl primer i7 and 10 μl 5×AmpliMix buffer. Amplifications were conducted using 20 cycles. PCR products were purified using NovoNGS® DNA Cleaning Beads.