GSM8698958: SN oocyte_oe-mCherry_KAS-seq_rep1; Mus musculus; OTHER
Label the oocytes with N3-Kethoxal, then extract the labeled genomic DNA using the BeyoMag™ Genomic DNA Small Sample Extraction Kit (Beyotime D0088S). KAS-seq was conducted following the published protocol60. Label the oocytes with N3-Kethoxal, then extract the labeled genomic DNA using the BeyoMag™ Genomic DNA Small Sample Extraction Kit (Beyotime D0088S). After labeling, fragment the DNA using Tn5 transposase (Vazyme TD501) under incubation conditions of 37 °C and 500 rpm for 30 min. Biotinylate the DNA using DBCO-PEG4-biotin (DMSO solution, Sigma 760,749). Next, purify the biotinylated DNA with the DNA Clean & Concentrator-5 kit (Zymo, D4013), retaining 5 μL of the labeled DNA as input, while the remaining 50 μL of DNA is used for enrichment. Wash 5 μL of Dynabeads™ MyOne™ Streptavidin C1 (Thermo 65,001) three times with 1 × binding and wash buffer. Then, resuspend the pre-washed beads in 2 × binding and wash buffer. Mix the beads with the 50 μL fragmented DNA obtained from the previous step and rotate gently at room temperature for 15 min. After incubation, place the mixture on a magnetic rack to remove the supernatant and wash the beads five times with 1 × binding and wash buffer. Elute the biotinylated fragments at 98 °C for more than 10 min. Perform PCR amplification using KAPA HiFi DNA polymerase; incubate the PCR reaction at 72 °C for 3 min, then at 98 °C for 45 s, followed by 14 cycles of amplification (15 s at 98 °C, 30 s at 60 °C, and 1 min at 72 °C).                                                                                      As previously described, ChIP-seq libraries for all samples were prepared using an ultra-low-input native ChIP protocol. Samples were thawed on ice and permeabilized in PBS containing 0.1% Triton-X-100 and 0.1% sodium deoxycholate. Micrococcal nuclease digestion was performed using 200 U of micrococcal nuclease (NEB, M0247S) in a customized digestion buffer. Samples were digested at 21°C for 7.5 min, and the reaction was terminated with 100 mM EDTA. Chromatin samples were pre-cleared with protein A/G beads in complete immunoprecipitation buffer, rotating at 4°C for 2 h, while antibodies were conjugated to beads at 4°C for 3 h. For each immunoprecipitation of oocytes, anti-H3K4me3 or anti-H3K36me3 antibodies were used. Chromatin was added to the antibody-bound beads, and samples were rotated at 4°C overnight. Samples were then subjected to two low-salt washes and one high-salt wash, followed by DNA elution from the beads at 65°C for 1.5 h. DNA was extracted and purified using phenol, chloroform, and isoamyl alcohol. And libraries were constructed using the Vazyme TD607 kit according to the manufacturer's instructions.