title: GSM8018737: flow; Homo sapiens; RNA-Seq
library_construction_protocol: For RNA single cell sequencing, chips were washed with PBS. To ensure that only cells growing in the hydrogel were collected, inlet and outlet ports were blocked with 3% agarose. After solidification of the agarose, cells were treated with a 1:1 mix of Collagenase P (18 U final concentration, Roche 11213857001) and Accutase and incubated for 30 min at 37°C. Pipette tips and tubes were washed with PBS + 2% BSA before usage to reduce cell loss. Successfully dissolved hydrogels and a single cell suspension was confirmed under the microscope before harvesting the cells in PBS + 10% FBS. The cells were centrifuged at 300 x g for 3 min, washed with PBS + 2% BSA and filtered with a 20 µm cell Pluristrainer (pluriSelect, 43-50020-023). Cells were counted with TrypanBlue (Sigma) and a cell vitality of over 70% was confirmed. RNA libraries were generated using Chromium Single Cell 3' library and gel bead kit v3.1 (10x Genomics). The amplified cDNA library was sequenced on a NovaSeq SP flow cell from Illumina.