title: GSM8966238: multiplexed single cell pool, 3x control, 4x KO [HTO]; Mus musculus; RNA-Seq
library_construction_protocol: Mice were euthanized using CO2 inhalation, and a gentle perfusion was performed to remove blood from the heart, after which the heart was collected in ice-cold HBSS. The tissue digestion was performed by utilizing the digestion mixture of dispase (1.2 units/ml), collagenase II (2mg/ml), and elastase (0.02 mg/ml) in a serum-free DMEM. A single cell suspension was produced with the help of debris removal solution (Miltenyi, 130-109-398) and RBC lysis buffer (Miltenyi, 130-094-183). The cell suspension was washed three times with PBS and re-suspended in MACS buffer (D-PBS + 2 mM EDTA + 0.5% BSA; filter sterile). 1 μg of single-cell hashing antibody was added to 100 μL of cell suspension (in staining buffer- 2% BSA/0.02% Tween), and incubated for 30 minutes at 4˚C. This was diluted with 2 mL of complete serum-free DMEM/PBS (v/v 2:1). FACS sorting was setup with live/dead exclusion and doublet discrimination, with a 1.5 mL collection tube containing 0.5 ml serum-free DMEM/PBS (v/v 2:1), Cells were stained with calcein-AM dye (Biolegend, 425201) and DAPI, which was incubated for 30 min on ice. Live single cells from individual mice with different hashtags were sorted into one collection tube, serving as one pooled sample for sequencing. Single live calcein AM-positive, DAPI-negative cells were sorted on the BD FACSMelody cell sorter (BD Bioscience) for single-cell RNA sequencing. The cell suspensions were counted with a Moxi cell counter and diluted according to the manufacturer's protocol to obtain 10.000 single-cell data points per sample. Each sample was run separately on a lane in Chromium controller with Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1 (10xGenomics). Single-cell RNAseq library preparation was done using a standard protocol.