GSM9084902: Lung organoid d19; Mus musculus; RNA-Seq
Single-cell suspension preparation. Discard the culture medium of BLOs, wash twice with PBS, and aspirated BLOs into a 6cm dish, using insulin syringe needle (328421) to remove matrigel from BLOs. The BLOs were digested by preheated 10 U/mL Papain (Worthington, LS003126) at 37 °C for 20 min in the incubator. Once the digestion was completed, an equivalent 10% FBS was added for termination. Cell suspensions were filtered through a 40-μm strainer (Falcon, 352340) and collected into a new LoBind tube on ice. After centrifugation at 300× g for 5 min at 4 °C, removed supernatant and resuspended with PBS plus 0.04% BSA to prepare for scRNA-seq library construction. Meanwhile, 0.04% trypan blue was introduced to determine cell viability via cell staining. Single-cell RNA library preparation and sequencing. Single-cell suspensions from each sample were loaded onto 10× Genomics Chromium v3.1 system to generate single-cell gel beads-in-emulsion (GEMs), where all generated full-length cDNA share a common 10× barcode. After incubation, GEMs were disrupted and cDNA was amplified via PCR. The single-cell 3' gene expression libraries were constructed using 10 μL cDNA and purified with SPRIselect. Libraries were quality controlled by Qsep100 for sized distribution and by Qubit 4.0 fluorometer for concentration quantification. Finally, sequencing was Illumina NovaSeq X Plus Sequencing,which provided by ANNOROAD GENE TECHNOLOGY.