title: GSM8098546: WT-H3K4me3-rep2; Mus musculus; OTHER
library_construction_protocol: In brief, approximately 1x105 single cells were carefully pipetted twice into a wash buffer. The pelleted cells were resuspended in wash buffer and activated concanavalin (Bangs Laboratories, BP531) and incubated for 15 minutes at room temperature. Cells bound to the beads were resuspended in a Dig-Wash Buffer and a 1:50 dilution of the primary antibody (rabbit anti-H3K4me3, Active Motif,39016; rabbit anti-Cxxc1, abcam, ab198977; normal IgG, Cell Signaling, 2729), and incubated at 4℃ overnight. The beads were then incubated with a secondary antibody (goat anti-rabbit IgG, SAB3700883, Sigma-Aldrish) diluted 1:100 in Dig-Wash buffer for 60 minutes at room temperature. Cells were treated with Hyperactive pG-Tn5 Transposase for CUT&Tag (Vazyme, S602) diluted in Dig-300 Buffer for 1 hour at room temperature. The cells were then resuspended in Tagmentation buffer (10mM MgCl2 in Dig-300 Buffer) and incubated at 37℃ for 1h. To stop tagmentation, 10l was spiked with 0.5M EDTA, 3l with 10%SDS, and 3l with 20mg/ml Proteinase K and incubated at 55℃ for 1h. DNA library amplification was performed according to the instructions and purified using VAHTS DNA Clean Beads (Vazyme, N411). Libraries were sequenced on an Illumina NovaSeq platform from Annoroad Gene Technology.