GSM6706399
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 total amount of 1 g DNA per sample was used as input material for the DNA library preparations. SQK-LSK109 Kit (Nanopore, UK) was used to construct 1D library. The DNA library was constructed by standard ligation method without DNA fragmentation, after end repaired, added the sequencing adaptor, motor protein and tether protein were connected to prepare the DNA library. After that, each amplicon fragment library was loaded into 1 R9 flow cell and sequenced on PromethION HAC (high accuracy) model.