GSM8079273: human_CD8_T_cell;N701_i7_2_N503_i5_2; Homo sapiens; OTHER
E14 mouse embryonic stem cells (mES cells) were cultured on gelatin-coated plates in DMEM supplemented with 15% FBS, 2 mM L-glutamine, 1% non-essential amino acids, 1% penicillin/streptavidin, 0.1 mM β-mercaptoethanol, 1,000 units/ml LIF (leukaemia inhibitory factor), 1 μM PD0325901, and 3 μM CHIR99021. The cells were maintained at 37°C and 5% CO2. Before cell sorting, the cells were harvested by centrifugation and passed through a 40 μm cell strainer (Falcon) to achieve single cell suspension. Optimized TAPS and CAPS+ reactions: For scTAPS, 96 cells were pooled for DNA purification and subjected to mTET1 oxidation as previously described11. A reaction mixture of 50 μl was prepared, comprising 50 mM HEPES buffer (pH 8.0), 100 μM ammonium iron (II) sulfate, 1 mM α-ketoglutarate, 2 mM L-ascorbic acid, 2.5 mM DTT, 1.2 mM ATP, 100 mM NaCl, and 4 μM mTet1CD. The reaction was conducted at 37°C for 80 minutes. Subsequently, 2.0 U of Proteinase K (New England Biolabs) was introduced to the reaction mixture and incubated at 50°C for 1 hour to stop the oxidation process. The oxidized DNA was purified using 1.8x AMPure XP beads, following the provided guidelines. To ensure complete oxidation, a second round of oxidation was performed. The double-oxidized DNA was purified and then underwent borane reduction under optimized conditions and purification using a Zymo column with Oligo Binding Buffer. For scCAPS+, 96 cells were pooled for DNA purification and subjected to a series of chemical reactions to convert 5hmC into 5fC and then to 5caC as previously described with some modifications12. DNA were incubated with 4-acetamido-2,2,6,6- tetramethylpiperidine-1-oxoammonium tetra-fluoroborate (ACT+ BF4-) in a 25 μl solution comprising sodium phosphate buffer at 25°C for 16 hours. The resultant oxidized DNA was purified using 1.8× AMPure XP Beads. then Pinnick oxidation is conducted in a 30 μl reaction mixture containing sodium acetate buffer, NaClO2, and 2- methyl-2-butene at 25°C for 16 hours. The purified product then underwent borane reduction under optimized conditions and purification using a Zymo column with Oligo Binding Buffer.  scTAPS and scCAP+ library construction and sequencing: Individual cells were isolated into a 96-well plate through FACS, each well containing 4 μL of lysis buffer consisting of 16.67mM TAPS-NaOH buffer (pH 8.5, Alfa Aesar, Cat: J63268), 8.33mM MgCl2(Invitrogen), 0.67x NEB buffer 4 (NEB, B7004S), 0.13%  Triton X-100 (sigma, X100-500ML), and 1 μg Qiagen protease. Following centrifugation at 2,000 rpm for 5 minutes, the plates were either preserved at -80°C or progressed to subsequent procedures. Single cell was lysate by incubating at 50°C for 3 h, followed by incubation at 75°C for 30 min, 80°C for 15 min, and a 4°C hold. Tagmentation was performed at 50°C for 15 minutes in a reaction mix consisting of 1x TAPS buffer (10mM TAPS-NaOH, 5mM MgCl2), 6% PEG (polyethylene glycol) 3,350, 1ul 1x preassembled Tn5, and H2O to a total volume of 10 μ. Tn5 was stripped off by adding 1.11 μL of 1.0% SDS (Sodium Dodecyl Sulfate), followed by incubating at room temperature for 5 minutes. Neutralization was performed by adding 1.24 μL of 5.0% NP40 or Triton X-100, followed by incubating at room temperature for 5 minutes. Subsequently, 12.35 μL of 2x Phusion High-Fidelity PCR Master Mix with HF Buffer (Thermo Scientific, F531L) was added, followed by incubating at 72°C for 5 minutes, and a 4°C hold. The 96 reactions were combined into a 50-mL BD tube and supplemented with 2-kb filler DNA (200 ng) and 0.1% spike in DNAs. Following purification, the TAPS or CAPS+ reaction, was employed as mentioned, and the final sequencing library was amplified utilizing the KAPA HiFi HotStart Uracil+ ReadyMix PCR Kit along with index primers from the Nextera XT Index Kit (Illumina, FC-131- 1001). The amplification protocol involved an initial step at 98°C for 45 seconds, followed by 13 cycles (98°C for 10 seconds, 60°C for 15 seconds, and 72°C for 1 minute), then incubation at 72°C for 3 minutes, 4°C hold. The library was subjected to purification and size selected to 500-700bp by 0.5x-0.25x Ampure XP beads. Custom  sequencing primers are used for custom Nova Sequencing (Read 1 Sequencing Primer: GCGATCGAGGACGGCAGATGTGTATAAGAGACAG; Read 2 Sequencing Primer: CACCGTCTCCGCCTCAGATGTGTATAAGAGACAG; Index 1 Sequencing Primer: CTGTCTCTTATACACATCTGAGGCGGAGACGGTG; Index 2 sequencing primer: CTGTCTCTTATACACATCTGCCGTCCTCGATCGC).