GSM8830132: FFPE MouseSpleen; Mus musculus; ATAC-seq
Tagmentation and combinatorial indexing via ligation 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. Reverse-crosslinking, gap filling and in vitro transcription After the third ligation, reverse cross-linking buffer (50 mM Tris-HCl, pH 8, 250 mM NaCl, 1 mM EDTA, and 1% SDS) and 0.22 mg/mL proteinase K were added to each tube, following previous publications 42, 43. The reaction mixture was incubated in a thermomixer at 1,200 rpm overnight at 65°C. The next day, a second proteinase K digestion was performed at 37°C for 2 hours at 850 rpm to ensure complete protein digestion. DNA was then purified using the Zymo ChIP DNA Clean & Concentrator Kit, following the manufacturer's instructions. For gap filling, an equal volume of NEBNext High-Fidelity 2X PCR Master Mix was added to the eluted DNA, and the reaction mixture was incubated at 72°C for 8 minutes in a thermal cycler. The sample was then purified using the Qiagen MinElute PCR Purification Kit, following the manufacturer's instructions. Further DNA purification was carried out using the 1X SPRI beads method to remove fragments shorter than 150 bp. Finally, the DNA was eluted in nuclease-free water. For in vitro transcription (IVT), a T7 RNA synthesis kit (New England Biolabs) was used. The following reagents were mixed: - 12.4 μL of template DNA - 2.5 μL of 10X T7 buffer - 1 μL of DTT - 2 μL each of ATP, CTP, UTP, and GTP - 1 μL of T7 mix - μL of RNase inhibitor (Thermo Fisher Scientific) The IVT mixture was incubated at 37°C overnight (16 hours), followed by DNase treatment and RNA purification using the ZYMO RNA Purification Kit (RNA Clean & Concentrator-5), according to the manufacturer's instructions. Finally, RNA was eluted in 20 μL of nuclease-free water. The RNA concentration was measured using NanoDrop, and the sample was either immediately used for DNA library preparation or stored at −80°C for future use. 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 The reaction is gently mixed and incubated at 42°C for 60 minutes and then at 70°C for 15 minutes. Next, 2.2 μL of 10× RNase H buffer and 0.2 μL of RNase H (5 U/μL) are added to each reaction and incubated at 37°C for 20 minutes. The RNAClean XP beads (1.8X) are used to purify the cDNA reaction mixture, and the cDNA is eluted in Qiagen Elution Buffer. A sequencing library mix is prepared using purified cDNA, NEBNext high-fidelity 2× PCR master mix, and a unique forward (i5) and reverse (i7) primer combination (DNA oligo sequences are provided in Supplementary Table 1). PCR amplification is run with the following cycling program: - 98°C for 30 seconds - 12 cycles of: - 98°C for 20 seconds - 63°C for 20 seconds - 72°C for 1 minute The sample is purified using the Qiagen MinElute PCR Purification Kit and run on a 6% PAGE gel. The gel region corresponding to 250–800 bp is cut, and the gel is made into fine pieces, which are incubated overnight at 55°C in crush-soak buffer (500 mM NaCl, 1 mM EDTA, 0.5% SDS) at 1,200 rpm. The DNA is purified from the gel using Costar spin-X centrifuge tubes and the Zymo ChIP DNA Clean & Concentrator Kit, following the manufacturer's instructions. A high-sensitivity DNA bioanalyzer chip is used to check the DNA size distribution and quantify the DNA concentration of the DNA library. Finally, deep sequencing is performed with NovaSeq  6000 on the mouse and human scFFPE-ATAC samples for single-cell analysis.