GSM9370527: HT1080 Control; Homo sapiens; RNA-Seq
At this time, media were aspirated, and collagen hydrogels were gently removed from their well and transferred to a 1.7ml tube. Using small surgical scissors, gels were cut into pieces before adding sterilized digestion buffer (2mg/ml Type 7 Collagenase (Worthington) in HBSS) at a 2:1 buffer to gel ratio. Samples were incubated at 37°C for 10 minutes before lightly vortexing and pipetting the samples. After an additional 5-10 minutes, when collagen was no longer visible, cells were pelleted in a microcentrifuge at 300g. Digestion buffer was aspirated, and the isolated cells were resuspended in 100ul trypsin (0.25%) and incubated at 37°C for an additional three minutes before 1ml of complete media was added to inhibit proteolysis. Samples were filtered into a 5ml falcon tube with a cell strainer cap before being pelleted by centrifugation (300g). Media was aspirated and cells were resuspended in 50µl FACS buffer (PBS + 0.5% BSA) for sequencing. Prepared single-cell suspensions were used for library preparation with 10X Genomics Chromium Single-Cell 3' v3 Chemistry following manufacturer's protocols.