GSM4503468: T-5405; Mus musculus; ATAC-seq
Thymus tissues isolated from 6- to 8-week-old male mice were gently ground in 1 mL of RPMI-1640. Thymocytes in a single-cell suspension were counted after passing through a 40 μm nylon mesh. A total of 1 × 106 thymocytes were stained with PerCP-Cy5.5-anti-CD45, PE-anti-CD8a and APC-Cy7-anti-CD4 antibodies (Biolegend) and then fixed in 1× PBS containing 1% methanal at room temperature for 5 min. After washing twice with 1× PBS, the cells were counted again. A total of 1 × 105 fixed cells were resuspended in 40 μL of 1× TD buffer (5 mM Tris-HCl, pH 8.0, 5 mM MgCl2 , and 10% DMF) containing 0.1% NP-40. Then, 10 μL of fluorescent transposomes were added and mixed gently. Fluorescent tagmentation was conducted at 55 °C for 30 min and stopped by adding 200 µL of 100 mM EDTA directly to the reaction mixture. The cells were loaded on a Sony SH800S sorter, and single cells of the CD45+/AF488-Tn5hi population were index-sorted into each well of 384-well plates. The 384-well plates used to acquire sorted cells were loaded with 2 µL of release buffer (50 mM EDTA, 0.02% SDS) before use. After sorting, the cells in the wells were incubated for 1 min. Plates that were not processed immediately were preserved at -80 °C. To prepare a single-cell ATAC-seq library, plates containing fluorescently tagmented cells were incubated at 55 °C for 30 min. Then, 4.2 μL of PCR round 1 buffer (1 μL of 100 μM MgCl2, 3 μL of 2× I-5 PCR mix [MCLAB], and 0.1 μL each of 10 μM R1 and R2 primers) were added to each well, followed by PCR: 72 °C for 10 min; 98 °C for 3 min; 10 cycles of 98 °C for 10 s, 63 °C for 30 s and 72 °C for 1 min; 72 °C for 3 min; and holding at 4 °C. Thereafter, each well received 4 µL of PCR round 2 buffer (2 μL of I-5 PCR Mix, 0.5 μL each of Ad1 and barcoded Ad2 primers, and 1 μL of ddH2O), and final PCR amplification was carried out: 98 °C for 3 min; 12 cycles of 98 °C for 10 s, 63 °C for 30 s and 72 °C for 1 min; 72 °C for 3 min; and holding at 4 °C. Wells containing different Ad2 barcodes were collected together and purified with a QIAquick PCR purification kit (Qiagen).