GSM9198841: Mouse spleen 50 UL reaction system scFFPE-ATAC; Mus musculus; ATAC-seq
Before tagmentation, 50,000 purified FFPE single-cell nuclei were centrifuged at 2,000 g for 5 minutes at 4°C and resuspended in 0.1% lysis buffer (10 mM Tris-Cl, pH 7.4, 10 mM NaCl, 3 mM MgCl₂, 0.1% IGEPAL CA-630). The nuclei were then centrifuged again at 2,000 g for 5–10 minutes at 4°C. The nuclei pellet was resuspended in 95 μl of 1× tagmentation buffer (10 mM Tris-HCl, pH 7.5, 5 mM MgCl₂, 10% dimethylformamide, and dH₂O), and 5 μl of barcoded FFPE-Tn5 transposase (2 mM) was added per sample. The reaction mix was gently mixed and incubated at 37°C for 30 minutes at 400 rpm. The reaction was stopped by directly adding EDTA to a final concentration of 30 mM to each sample. All samples were then pooled and centrifuged to obtain a nuclei pellet. Next, three rounds of ligation were performed using the split-and-pool technique in a 96-well plate, with each well containing a ramped specific linker and ligation barcode. Detailed DNA sequences are provided in Supplementary Table 1. In brief, the ligation process was performed as follows: the pooled nuclei were resuspended in 1 mL of 1× NEBuffer 3.1 and mixed with the ligation mix (100 μL of 10× NEBuffer 3.1, 22 μL of 50 mg/mL BSA, 500 μL of 10× T4 DNA ligase buffer, 2,278 μL of ultrapure water, and 100 μL of T4 DNA ligase). A total of 40 μL of this mix was transferred per well, containing 10 μL of the ramped Ligation1 mixture, and incubated at 37°C for 30 minutes at 400 rpm. Then, 10 μL of Blocker-Ligation1 solution (2.64 μL of 100 μM Blocker-Ligation1, 2.50 μL of 10× T4 ligation buffer, and 4.86 μL of ultrapure water) was added per well, and the reactions were incubated at 37°C for 20 minutes at 400 rpm. The samples were then pooled into a 15 mL tube pre-coated with 0.5% BSA, followed by centrifugation at 1,500 g for 10 minutes at 4°C. The second and third ligation reactions were performed identically to the first. After the second and third ligations, blocking reactions were carried out using: 10 μL of Blocker-Ligation2 solution per well (2.64 μL of 100 μM Blocker-Ligation2, 2.50 μL of 10× T4 ligation buffer, and 4.86 μL of ultrapure water); 7.5 μL of Terminator_Ligation3 solution per well (2.64 μL of 100 μM Terminator_Ligation3, 2.50 μL of 0.5 M EDTA, and 2.36 μL of ultrapure water) for reaction termination. After gently mixing, the samples were pooled again into a 15 mL tube pre-coated with 0.5% BSA, followed by centrifugation at 1,500 g for 10 minutes at 4°C. After the third ligation, each nucleus acquired a unique combination of barcodes. Finally, the pellet was resuspended in PBS, nuclei were counted, and the sample was split into tubes containing 30,000–80,000 nuclei for reverse crosslinking. Single cell FFPE-ATAC DNA library preparation Single-stranded cDNAs are prepared from 0.5–1 μg of purified IVT transcripts using the FirstStrand_cDNA oligo (DNA oligo sequences are provided in Supplementary Table 1). The reaction mixture (11.25 μL), containing 500ng-1 μg of RNA, 1 μL of 100 μM random octamer primer stock, and 0.25 μL of RNase Inhibitor (20 U/μL), is heated at 70°C for 3 minutes, then immediately cooled on ice. Next, the master mix is added, which consists of: • 4 μL of 5× First-Strand Buffer • 2 μL of dNTP Mix • 2 μL of 100 mM DTT • 0.25 μL of RNase Inhibitor (20 U/μL) • 0.5 μL SMART MMLV RT