GSM7500656: MALBAC_GM12878_rep3; Homo sapiens; OTHER
Resuspended ligated nuclei pellets in 200μL 0.25%BSA in PBS each tube and added 0.2μL 1000×DAPI for sorting nuclei. Single nucleus was flow sorted into each well of 96-well plate containing 2 μL lysis buffer(0.04 μL 1M Tris-HCl (pH 8.0),0.08μL 500 mM NaCl,0.03μL10% Triton X-100 (Sigma), 0.0004μL 0.5 M EDTA,0.05μL 20mg/mL QIAGEN protease). And then to decrosslinking and digest, incubated at 50°C, 3h; 70°C, 20 min; 68°C, 45 min; 4°C pause. After digesting, 96-well plates were stored at −80 ˚C. A single cell was flow sorted into each well of 96-well plate containing 2.5 μL lysis buffer (0.25 μL 100 mM Tris-EDTA, 0.075μL 10% Triton X-100, 0.05μL 1 M KCL, 0.125μL 20mg/mL QIAGEN protease). And then incubated at 50°C for 3h; inactivate the protease at 70℃ for 30 min; 4°C pause. After digestion, we added 15μl amplification mixture (1.5μL 10×Thermopol buffer, 0.6μL 2.5 mM dNTP, 0.2μL 100 mM MgSO4, 0.5μL 10 μM GAT-N5-3G primer, 0.5μL 10 μM GAT-N5-3T primer, 0.5μL 2 U/μl DeepVentR (exo-) polymerase; New England BioLabs, M0259L ) into each tube containing lysed single cell, and performed quasilinear pre-amplification: 95℃, 3 min; then 11 cycles of 4°C for the 50s, 10°C for 50s, 20°C for 50s, 30°C for 50s, 40°C for 45s, 50°C for 45s, 65°C for 4min, 95°C for 20s, 58°C for 20s. And then added 15μl similar amplification mixture within GAT-8nt-barcode-primer, the products were then exponentially amplified for 12 cycles of 95°C for the 20s, 58°C for 30s,72°C for 3min, and finally 72°C for 5 minutes. After amplification, we pooled products with different barcodes together and then purified them with DNA Clean & concentration-5 kit (Zymo Research, D4014) with 5× binding buffer. cDNA products were further used to construct of libraries for next-generation sequencing as the previous report