GSM5901231: day_9_1_3D_suspension_culture_; Homo sapiens; RNA-Seq
Matrix-free samples were washed with PBS, resuspended in Accutase and incubated for cell detachment at 37°C for 30 min. The reaction was stopped by adding five volumes of media. Afterward, cells were washed once with PBS. Prior to this process, Matrigel embedded samples were washed with PBS, incubated with 0.5 mL Collagenase/Dispase Solution (1:100 to a final concentration of 1 mg/mL in StemPro34) for around 4 h, until organoids detached from the Matrigel. The enzymatic reaction was stopped with 1 mL Neutralisation Buffer (1%BSA, 1%P/S in DMEM:F12). The single cells were cryo-preserved in DMEM with 10% heat-inactivated FBS (Thermo Fisher Scientific) and 10% DMSO based on a previously described scRNA-seq sample preparation protocol(Guillaumet-Adkins et al., 2017). For sequencing, cryo-preserved cells were thawed in DMEM:F12. 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 6000 S2 flow cell from Illumina.