GSM8730175: Mouse round spermatids, CTCF, CUTNRUN, rep2; Mus musculus; OTHER
Round Spermatids were separated from testis of adult C57B6J mice (8 weeks), using counterflow centrifugal elutriation (CCE) methods. For ATAC-seq, 1 × 10^4 Round spermatids were sorted and were incubated with 10 μl 5 × TTBL, 5 μl TTE Mix V50 in a 1.5 ml microtube at 55 °C for 10 min for fragmentation. The fragment products were purified using VAHTS DNA Clean Beads. CUT&Tag experiments were performed with Hyperactive Universal CUT&Tag Assay Kit for Illumina Pro (Vazyme Biotech, TD904). Briefly, 2 × 10^4 round spermatids were sorted and washed once in 0.5 ml wash buffer, and were incubated with 10 μl pre-activated concanavalin ConA beads in a 1.5 ml microtube at 25 °C for 10 min. H3K4me3 (07-473, Milipore), H3K27me3 (c36811, CST) or CTCF (07-729, Milipore) antibody buffer at 1:100 dilution was added and cultured with cells at 4°C overnight. Cell were washed with dig-wash buffer and incubated with secondary antibodies at 25°C for 1h. After washing thrice with dig-wash buffer, 100ul pA/G-Tnp in dig-300 buffer were added to samples and then rotated at 25 °C for 1 h. After washing, cells were resuspended in 40 μl dig-300 buffer with 10 ul 5 × TTBL and incubates at 37 °C for 1 h. 5 μl Proteinase K, 100 μl Buffer L/B and 20 μl DNA Extract Beads were added to samples and incubated at 55 °C for 10 min. CUT&RUN experiments were performed with Hyperactive pG-MNase CUT&RUN Assay Kit for Illumina (Vazyme Biotech, HD102). 2 × 10^4 Round spermatids were sorted and washed once in 0.5 ml wash buffer, and were incubated with 10 μl pre-activated concanavalin ConA beads Pro in a 1.5 ml microtube at 25 °C for 10 min. Cells were incubated with 1:100 dilution antibody buffer of H3K4me3 (07-473, Milipore), H3K27me3 (c36811, CST) or CTCF (07-729, Milipore) antibody buffer at 4°C overnight. After washing, 100ul pG-MNase Enzyme premix were added to samples at 4 °C for 1 h. After washing, 100ul CaCl2 premix were added to samples and then incubate on ice for 60-90 minutes. 100 μl Stop Buffer were added to samples and then incubate at 37 °C for 10-30 minutes and then extract DNA. ATAC-seq libraries were constructed using TruePrep DNA Library Prep Kit V2 for Illumina (Vazyme, TD501). CUT&Tag libraries were constructed using Hyperactive ® Universal CUT&Tag Assay Kit for Illumina (Vazyme, TD903). CUT&RUN libraries were constructed using Hyperactive pG-MNase CUT&RUN Assay Kit for Illumina (Vazyme, HD102). All libraries were sequenced on the Illumina NoveSeq 6000 platform to generate 150 bp pair-end reads.