title: Establishing and optimizing the C4_mtscATAC-seq protocol in a microfluidic platform
design: The frozen cells of HEK-293T and 3T3-L1 were resuscitated quickly in a constant temperature water bath of 37C with gent shaking. After resuscitating fully, the cells were centrifuged at 500 g, 5 min, 4. The cell pellets were resuspended in 1 mL DMEM and stained with trypan blue to determine the proportion of viable cells. Only the cell samples with more than 90% cell viability rate will be chosen for C4_mtscATAC-seq. The cells with high quality (> 90% cell viability rate) were resuspended in 1mL precooled absolute methanol, and incubated on ice for 10 min. After centrifugation at 4, 300 g for 5 min, the supernatant was discarded. The cell pellets were resuspended gently in 1ml pre-cooled PBS, and centrifugation was conducted again at 4, 300 g for 5 min to remove the supernatant. The cell pellets were gently resuspended in 100 L Lysis Buffer A Tris-HCl (10 mM, pH=7.5), NaCl (10 mM), MgCl2 (3 mM), NF-H2O, BSA (1%) in PBS and NP40 (0.1%) or Tween-20 (0.05%), and incubated on ice for 5 min. Then 1 mL Wash Buffer A was added in the cell suspensions, and centrifugation was done at 4 500 g for 5 min. After discarding the supernatant, the cell pellets were additionally washed with 400 L Wash Buffer A containing Tris-HCl (10 mM, pH=7.5), NaCl (10 mM), MgCl2 (3 mM), NF-H2O, BSA (1%) in PBS. The cells were resuspended in PBS containing 1% BSA gently and stained with DAPI for counting the cell number. A total of 100000 cells were transposed for each type of cell. The transposition system was 50 L containing 4 L transposase (Tn5). Transposition was conducted in PCR instrument, and 5000 transposed HEK-293T and 5000 transposed 3T3-L1 were mixed and used for droplet generation.