GSM8329920
Kidney organoids were dissociated to single cell, incubated with Totalseq-A antibodies (Biolegend; Cat# 394601, 394603, 394605, 394607, 394609, 394611, 394613, 394615) for sample hashing each samples collection day, pooled and super-loaded onto the 10x Genomics Chromium Controller (10x Genomics) to capture single cells. Briefly, 3x technical replicates for each sample were dissociated using TrypLE for 3 minutes, pooled and diluted in TEsR-E6 and centrifuged at 300 × g for three minutes. The cell pellets were resuspended in 100 µL cold Fluorescence-Activated Cell Sorting (FACS) buffer (PBS with 2% fetal bovine serum). Then, 2 µL of Totalseq-A hashing antibody was added and the cells were gently pipetted to mix before incubating for 20 min on ice. Cells were then washed twice with FACS buffer by centrifuging at 300 × g for 5 min, discarding the supernatant and resuspending the cell pellet in 100 µL cold FACS buffer. Approximately 40,000 cells were loaded onto the Chromium Single Cell Chip B (10x Genomics; Catalog Number: PN-1000073) to capture 20,000 single cells with the Gel Bead Kit V3.0 (10x Genomics; Catalog Number: PN-1000075). GEM generation, barcoding, cDNA amplification, and library construction were performed according to the 10x Genomics Chromium User Guide (CG000183). Libraries were prepared with the Single Cell 3' V3.0 Library and Gel Bead Kit (10x Genomics; Catalog Number: PN-1000075). The samples collected from stages 1, 2 and 3 occurred across six days, each capture multiplexed up to eight unique conditions and all were sequenced on the same run. The final collection of stage four cells occurred separately, multiplexing either 3 or 4 conditions to allow for greater cell numbers per condition for the more complex “mature” organoids.