GSM7848803: EP cells H3K4me3_CUT_Tag rep 1; Mus musculus; OTHER
P0 mice were euthanized and the back skin was carefully removed using scissors and forceps. The extracted back skin was then placed into a culture dish with the dermal side facing downwards. Next, to separate the epidermis from the dermis, the dispase II was used to incubate these skin tissues overnight at 4°C. The following day, the skin was transferred to a new culture dish and the epidermis was carefully separated from the dermis using forceps. Subsequently, the separated epidermis was finely diced and incubated with 0.25% trypsin solution at 37 °C for 5 minutes. The digested solution was then filtered and subjected to centrifugation at 500g for 5 minutes at 4 °C, leading to the enrichment of primary epidermal keratinocytes in the resultant cell pellet. We further used fluorescence-activated cell sorting (FACS) to isolate cells that specifically express integrin α6 (CD49f), which were progenitor cells of the epidermis and hair placodes/germs. NovoNGS High-Sensitivity CUT&Tag 3.0 Kit is used to extract DNA fragments. We constructed the libraries using the NovoNGS High-Sensitivity CUT&Tag 3.0 Kit (Novoprotein, N259-YH01) according to the manufacturer's instructions. In Brief, epidermal basal layer cells (approximately 20,000 cells per sample) isolated by the above method were bound with Concanavalin A-coated beads. Then, they were incubated with primary antibody anti-H3K4me3 (1:50; Invitrogen, MA5-11199) for 2 hours at room temperature. The beads were washed several times with wash buffer and then incubated with secondary antibody goat anti-rabbit Immunoglobulin G (1:100; Invitrogen, 31210) for 1 hour at room temperature. After washing again several times, these beads were incubated with the ChiTag pAG-Tn5 transposon for 1 hour at room temperature. Then, tagmentation buffer (containing MgCl2) was added and incubated at 37°C for 1 hour, and this process was stopped by adding 10% SDS and incubating at 55°C for 10 minutes. The DNA fragments were then purified and extracted using Tagment DNA Extraction Beads. A 50 ul PCR amplification system was prepared: 33 μl DNA fragments, 2.5 μl i5 primer (10μM, N501), 2.5 μl i7 primer (10μM, N701 or N702) and 10 μl 5×mpliMix buffer. The number of cycles of amplification was determined by agarose gel electrophoresis.