GSM9590064: zebrafish 128 cell, PCR_EM-seq; Danio rerio; OTHER
HeLa cells were detached, collected, and pelleted by centrifugation at 300 × g for 5 min. Genomic DNA was subsequently extracted using the FastPure Blood/Cell/Tissue/Bacteria DNA Isolation Mini Kit (Vazyme) according to the manufacturer's protocol. Genomic DNA was fragmented to an average size of ~300  bp using a Covaris M220 Focused-ultrasonicator. Embryos of zebrafish were collected at four developmental stages: 64-cell, 128-cell, 1k-cell, and Germ ring. Before DNA extraction, embryos were bleached with bleach solution (0.1 ml 5% NaClO in 170 ml system water) for 5 min and rinsed thoroughly with 1× PBS. Genomic DNA was isolated using the FastPure Blood/Cell/Tissue/Bacteria DNA Isolation Mini Kit (Vazyme), according to the manufacturer's protocol. Genomic DNA from the embryos was fragmented to an average size of around 300 bp using the Bioruptor Pico sonication device (Diagenode). For the 128-cell, 1K-cell, and Germ-ring samples, an optimised sonication program was used, i.e., 15 s ON/30 s OFF, 4 cycles for the 50-μl sample volume. For the 64-cell-stage gDNA, 3 cycles of 15 s ON/30 s OFF were used instead. End repair and A-tailing were performed for fragmented DNA using the VANTS Universal DNA Library Prep Kit for Illumina V3 (Vazyme) with EM-seq adapters (NEB) according to the manufacturer's protocol. After adapter ligation, unmethylated cytosines in the DNA were enzymatically converted to uracil using the EpiArt DNA Enzymatic Methylation Kit (Vazyme), following the manufacturer's protocol. For FINE-EM-seq libraries, FINE amplification was applied on converted DNA(including 1 ng, 100 pg, 1 pg HeLa gDNA and 1 ng of each stage's zebrafish gDNA), followed by a 3-cycle PCR to add full-length adapters for Illumina sequencing. For PCR methylation libraries, converted DNA was amplified using 2× Synthesis Mix (ABclonal) or HiFi V3 (Vazyme). PCR thermal cycling consisted of an initial denaturation at 98°C for 45 s; followed by 16 cycles (for 1 ng), 20 cycles (for 100 pg), or 23 cycles (for 10 pg) of 98°C for 15 s, 60°C for 30 s, and 72°C for 20 s; followed by a final extension at 72°C for 1 min and then hold at 12°C. The amplified DNA was purified twice using 0.8× VANTS DNA Clean Beads (Vazyme).