single-cell ATAC-seq of Homo sapiens: male breast cancer tumor: Sample MAND2588_4CC61L-ATAC
Fresh, never-frozen tumor samples were minced with two sterile razor blades and incubated overnight on a 37°C stir plate at 180rpm in the following digestion media: DMEM/F12, 5% FBS, 15mM HEPES, 1x Glutamax, 1x Collagenase/Hyualuronidase, 1% penicillin/streptomycin, and 0.48 µg/mL Hydrocortisone. After overnight digestion, tumor cells were pelleted at 1200rpm for 5 min at room temperature and then washed two times with cold PBS + 2% FBS and 10mM HEPES (PBS-HF). Removal of red blood cells was done by treating cells with cold ammonium chloride solution diluted with 1 part PBS-HF. To ensure complete dissociation of tumor cells, cells were treated with 0.05% Trypsin-EDTA for 1 minute, followed by trypsin inactivation with PBS-HF and centrifugation. Next, a 1 minute treatment with Dispase and 1 mg/mL DNase I was done, followed by inactivation with PBS-HF and centrifugation. Cells were then washed with PBS-HF, filtered through a 100µm cell strainer and a final centrifugation was done. Cell pellets were resuspended in DMEM/F12 + 5% FBS using a volume that was dependent on the size of the final cell pellet, and then filtered through a 40µm cell strainer. Cell viability was measured with the Countess II Automated Cell Counter. For scRNA-seq libraries, 10,000 cells were used for library preparation using the 10x Genomics Single Cell 3' kits: Single Cell 3' GEM, Library & Gel Bead Kit v3 (PN-1000075), Chromium Chip B Single Cell Kit (PN-10000153), and Chromium i7 Multiplex Kit (PN-120262) following the manufacturer's protocol. For scATAC-seq, nuclei isolation of 500,000 cells was done following the Nuclei Isolation for Single Cell ATAC Sequencing protocol from 10x Genomics using a 4 minute lysis time. Next, 10,000 nuclei was used for library preparation using the 10x Genomics Single Cell ATAC Kits: Chromium Single Cell ATAC Library & Gel Bead Kit v1 (PN-1000110), Chromium Chip E Single Cell ATAC Kit (PN-1000082), and Chromium i7 Multiplex Kit N, Set A (PN-1000084) following the manufacturer's protocol. scRNA-seq and scATAC-seq libraries were sequenced using 10x Genomics' suggested sequencing parameters on an Illumina NextSeq 500 machine.