GSM7917922: Ac, 5fU-DIP-2; Amphidinium carterae; OTHER
Total RNA was extracted from A. carterae and Symbiodinium sp. using RNAzol®RT (MRC) according to the manufacturer's protocol.  Genomic DNA of A. carterae and Symbiodinium sp. were extracted via a CTAB method. Briefly, cell pellets were resuspended in 600 µl of preheated CTAB extraction buffer (100 mM Tris pH 8.0, 20 mM EDTA pH 8.0, 2 M NaCl, 2% (w/v) CTAB, 2% (w/v) Polyvinylpyrrolidone. Next, 10 µl of 20 mg/ml RNase A (NEB) was added, and the mixture was incubated at 37°C for 2 hours, followed by the addition of 20 µl of Proteinase K (NEB) and another incubation at 65°C overnight. Equal volumes of phenol: chloroform: isoamyl alcohol (25: 24: 1) (Solarbio Life Sciences, Beijing, China) were added. After centrifugation, the upper phase was transferred to a new Eppendorf tube, mixed with 2/3 volume of isopropanol, and then centrifuged at 13000 rpm for 10 minutes. The pellet was washed with 700 µl of 75% ethanol and 80% ethanol sequentially. Finally, the DNA was eluted in 20 µl of ddH2O. 1. A. carterae Genome Construction: gDNA extracted from A. carterae was sent to Beijing Genomics Institute (BGI, Shenzhen, China) for Pacbio HiFi and Hi-C library construction. 2. Transcriptome Sequencing: For short-read RNA-seq and long-read sequencing of A. carterae, isolated total RNA from A. carterae cells was sent to BGI for strand-specific mRNA library and PacBio library construction. 3. 2,4-Pyridinedicarboxylic Acid Treatment: Total RNA of A. carterae was treated with Ribo-off® rRNA depletion kit (N409, Vazyme Biotech, Nanjing, China) and sent to BGI or Novogene Company (Beijing, China) for stranded RNA-seq library construction. 4. 5hmU-DIP and 5fU-DIP Library Construction: A. carterae gDNA was sonicated, end-repaired, adapter-ligated using the VAHTS® Universal DNA Library Prep Kit for Illumina V3 (ND607, Vazyme Biotech, Nanjing, China), and subjected to 5hmU-DIP using anti-5hmU antibody and Protein A/G beads, followed by protease K digestion and DNA purification. For the parallel 5fU-DIP, ACT+BF4- oxidation was performed first to fully convert gDNA 5hmU into 5fU, with the subsequent steps identical to the 5hmU-DIP procedures. 5. 5hmU-Chemical-Seq: For the 5hmU-chemical-seq approach, we employed both commercial and modified adapters. The modified adapters were designed with a 5'-end amino group and a 3'-phosphorylated oligodeoxynucleotide, annealed to create final adapters 5N3A3 and 5N3A4. A. carterae gDNA was fragmented, end-repaired, and ligated to these pre-annealed adapters. After ligation, DNA purification was performed, followed by ACT+BF4- oxidation treatment. The oxidized DNA samples were then biotinylated, purified, and captured using streptavidin magnetic beads. After washing, the DNA was eluted and amplified for subsequent sequencing. For controls, non-oxidized DNA was prepared using commercial adapters and amplified using the VAHTS® Universal DNA Library Prep Kit for Illumina V3.  6. WGBS Library Construction: A. carterae gDNA was sent to Novogene Biotech Co., Ltd (China) for WGBS libraries construction according to a standard protocol. Briefly, gDNA was spiked with unmethylated lambda DNA and sheared to average 200 bp size, followed by bisulphite treatment using the Accel-NGS® Methyl-Seq DNA Library Kit (Swift Biosciences). Following this treatment, the DNA was subjected to end-repair, adapter ligation, and PCR amplification.