GSM8971657: single-cell D&D-seq, CA46 (CTCF) and K562 (GATA1) mixed; Homo sapiens; ATAC-seq
For all the following centrifugations, cells were centrifuged in a swing-bucket centrifuge at 300g for 5 minutes before fixation and 600g for 5 minutes after fixation. About 200K to 1 million cells were collected and resuspended in 400 µl PBS. Then, 16% methanol-free formaldehyde (Thermo Fisher Scientific, PI28906) was added for fixation (final concentration 0.1%) at room temperature for 5 minutes. Glycine (final concentration 125 mM) was added to stop the cross-linking, followed by a wash with 1 ml of PBS. Fixed cells were permeabilized in 200 µl permeabilization buffer (20 mM Tris-HCl pH 7.4, 150 mM NaCl, 3 mM MgCl2, 0.1% NP40, 0.1% Tween-20, 1% BSA, 1× protease inhibitors) on ice (7 minutes for cell lines, 5 minutes for primary cells), followed by a wash with 200 µl cold wash buffer (20 mM HEPES pH 7.6, 150 mM NaCl, 0.5 mM spermidine, 1% BSA, 1× protease inhibitors). After cell counting, 100K-400K cells were transferred to PCR tubes and resuspended in 100 µl antibody buffer (20 mM HEPES pH 7.6, 150 mM NaCl, 2 mM EDTA, 0.5 mM spermidine, 1% BSA, 1× protease inhibitors), with 1 µl (1:100 dilution) of the antibodies. The cells were incubated at 4 °C with slow rotation overnight. The cells were washed once with 200 ul wash buffer, and another time with 200 ul D&D binding buffer. The cells were resuspended in 50 ul D&D activation buffer with 100 uM DddA_NT, and incubated at room temperature on a rotator for 1 hour. The cells were then washed with 200 ul D&D binding buffer, resuspended in 50 ul DnD activation buffer, and incubated at 37 °C for 1 hour for deamination. After D&D activation, the cells were resuspended in 1x nuclei buffer and counted using trypan blue and a Countess II FL Automated Cell Counter. For the remaining steps, we follow single-cell ATAC-seq per 10x protocol (version CG000209 Rev F, 10x Genomics) with the following modifications: During the GEM generation and barcoding reaction (step 2.1), 2 ul of uracil-tolerant DNA polymerase (NEBNext Q5U Master Mix, NEB M0597S) was added to the barcoding reaction mixture to facilitate the first few rounds of PCR, where we expect to have some uridine in the reaction. Samples are sequenced PE150 or PE100.