library_construction_protocol: Single Cell Dissociation of 3D Organoids: Day 18 3D kidney organoid were harvested from Aggrewell HT into a 1.5 mL tube using a wide bore pipette and washed twice with PBS. 1 mL of cold Trypsin-EDTA (0.05%) and Dnase (1:50) were added and the organoids were incubated at 37°C for 10 minutes then triturated 2X with a P1000 pipette. The organoids were again incubated for another 10 minutes then triturated 5X to generate a single cell suspension. FACS buffer was added to the suspension to deactivate the trypsin. Single cells were centrifuged at 300g for 5 mins, then the supernatant was discarded and 1 mL of pre-warmed Cell Suspension Buffer was added. Cell concentration, viability and percent of clumps were determined using a Nucleocounter.  Single Cell Dissociation of 2D Organoids: On day 18, 2D monolayers were washed with PBS and kidney tubules were first enriched by a crude removal of the mesenchymal monolayer. This was done by incubating the monolayers at 37°C for 10 minutes after adding cold Trypsin-EDTA (0.05% and Dnase (1:50). Then with a P1000 pipette and medium force, the trypsin-Dnase mixture was sprayed onto the cells 5X to detach the mesenchymal cells. This trypsin-Dnase-single cell suspension mixture was removed from the well and discarded. Fresh trypsin and Dnase were added to the remaining tubules in the well and the plate was incubated at 37°C for 20 minutes. The tubules were then triturated 5X with a P1000 then incubated again for 5 minutes. A single cell suspension was generated after another round of 6X trituration and FACS buffer was added to the suspension to deactivate the trypsin. Single cells were centrifuged at 300g for 5 mins, then the supernatant was discarded and 1 mL of pre-warmed Cell Suspension Buffer was added. Cell concentration, viability and percent of clumps were determined using a Nucleocounter.  PIPseq V Cell Preparation: After the cell counts, both 2D and 3D samples were then centrifuged at 200g for 3 minutes (RT), followed by aspiration of the supernatant. 200-400uL of cold cell suspension buffer is added to the pellet and gently mixed 10-15 times to resuspend. The single cell concentration was determined using both the Nucleocounter and the Luna Fx7 Automated Cell Counter. A suspension of 2,500 live cells/uL is prepared in a cell suspension buffer to achieve an input volume of 4uL (10,000 live cells/uL) into a PIP reaction. Library preparation was performed according to the manufacturer's instructions (Illumina Single Cell 3′ RNA Prep, T10, Illumina). Briefly, single cells were combined with Particle-templated Instant Partitions (PIPs) and barcoded beads to generate emulsified partitions. Following chemical lysis, mRNA released from each partition was reverse transcribed into barcoded cDNA. The cDNA was then amplified by whole transcriptome amplification, isolated from PIPs, and purified using magnetic beads. Quality control included Qubit quantification and fragment analysis. For library construction, cDNA underwent fragmentation, end repair and A-tailing, adapter ligation, and sample index PCR to add Illumina P5, P7, and index sequences. Final libraries were cleaned with magnetic beads and prepared for sequencing on Illumina platforms.