GSM7899155: Pooled snRNA-seq Heart; Homo sapiens; RNA-Seq
For PBMC isolation blood was collected from patients into EDTA-tubes and mixed 1:1 with PBS. EDTA-blood was then carefully layered on to Ficoll-Paque Plus (1:1,5) and centrifuged (400 G, 40 min, RT). After centrifugation the PBMC-Layer was aspirated, resuspended in FACS-Buffer (PBS, 2% FCS, 2mM EDTA) and centrifuged (300 G, 10 min, RT). The supernatant was discarded and cells were resuspended in 300 µl FACS Buffer. At this step, PBMC were counted and ~0.5x106 cells were taken for each Bulk-RNA sequencing and WES. The remaining cells were stained with CD14-PE, CD16-APC, CD4-FITC, CD8-PE Cy7 and CD19-BB700 (30 min, 4°C, protected from light). Afterwards, cells were washed once with FACS Buffer (400 g, 5 min, 4°C) before staining PBMC with a unique CellPlex-Oligo (100 µl, 5 min, RT) for each patient. Subsequently cells were washed twice, before sorting a unique PBMC population for each patient (CD14 Monocytes, CD16 Monocytes, CD4 T-Cells, CD8 T-Cells, CD19 B-Cells) using a BD FACSMelody Cell Sorter. For lymphocytes Dapi, CD16 and CD14 positive cells were excluded to avoid dead cell and monocyte contamination. For monocytes Dapi, CD4 and CD8 positive cells were excluded to avoid dead cell and lymphocyte contamination. After sorting, PBMC were pooled and immediately loaded onto a Chromium Next GEM Chip G for snRNA-seq (10x, 3' v3.1). For heart samples, Snap frozen tissue was crushed using a mortar and pestle, resuspended in 500 µl nuclei lysis Buffer (NLB, EZ lysis Buffer NUC101, Sigma-Aldrich with 1 Tab/10 ml  of Complete Protease Inhibitor, Roche and 10 µl/ml Recombinant RNase Inhibitor 2313A, Takara Bio and 10µl/ml Superase In RNase Inhibitor, Thermofisher) and homogenized with dounce tissue grinder pestles. The homogenized solution was spun down, supernatant discarded and the pellet resuspended in 4 ml nuclei resuspension buffer (NRB, PBS, 1% BSA, 10 µl/ml Protector RNase Inhibitor, Rochel). The suspension was then filtered via a 40 µm cell strain and centrifuged (500 G, 4°C, 5 min). Supernatant was discarded, and cells resuspended in 200 µl Nuclei resuspension Buffer (NRB, PBS, 1% BSA, 10 µl/ml Protector RNase Inhibitor, Rochel) with 3 µl of a unique TotalSeq anti-Nuclear Pore Complex Antibody Hashtag-Antibody for each sample (4°C, 20 min). Subsequently samples were washed with 1 ml NRB, centrifuged (500 G, 4°C, 5 min) and resuspended in 100 µl of a unique CellPlex Oligo for each sample (RT, 5 min). Cells were washed once more with NRB (500 G, 4°C, 5 min) before proceeding to Fluorescent-activated Nuclei-sorting of DAPI positive nuclei with a Sony SH800S. 5 minutes prior to sorting nuclei were stained with DAPI (Sigma-Aldrich). After sorting, nuclei were pooled and immediately loaded onto a Chromium Next GEM Chip G for snRNA-seq (10x, 3' v3.1). Single cell RNA (3' v3.1, Dual Index) and 3' CellPlex library preparation was performed according to the manufacturer's instructions. Hashtag-Libraries were prepared as described by Stoeckius et al.