NextSeq 500 paired end sequencing; Single cell RNA-seq of hiPSC-derived kidney organoids differentiated within self-assembling peptide hydrogels and the animal-derived matrix Matrigel
Single cell RNA-seq of hiPSC-derived kidney organoids differentiated within self-assembling peptide hydrogels and the animal-derived matrix Matrigel
hiPSCs were differentiated using a modified version of the Takasato et al. 2016 protocol. Briefly, hiPSCs were differentiated as a cell monolayer using CHIR99021 and FGF9 treatments for 7 days to induce intermediate mesoderm. Cells were subsequently pelleted and grown in a suspension culture for 48 hours. Organoids were then encapsulated into either synthetic self-assembling peptide hydrogels of variable mechanical strengths or Matrigel and differentiated until day 24. 6 organoids per support matrix were then pooled and dissociated using the cold-active protease from Bacillus licheniformis for scRNA-seq experiments. Following dissociation, single cell suspensions were loaded onto the Chromium Next GEM Chip G (Cat number: 1000127) with reverse transcriptase master mix and Gel Bead Kit (Cat number: 1000128) to achieve cell recoveries of 3,000-4,000 cells. GEMs (gel beads in emulsion) were formed as per manufacturers instructions using the 10X Chromium Controller and Single-cell 3 v3 kits. Libraries were prepared using the Chromium Single Cell 3' v3 Library and Gel Bead Kit (Cat number: 1000128) and Single Index Kit T Set A (Cat number: 1000213) as per manufacturers (10X Genomics) instructions.