GSM6245110: differentiated ureteric bud lineages, day 7 (nephric duct) and day 18 (collecting duct); Homo sapiens; RNA-Seq
Organoids were isolated from matrigel culture by incubation in Cell Recovery Solution (Corning) on ice for 15-20 minutes, with gentle trituration using a wide-bore P1000 pipet every 3-4 minutes. Once separated from Matrigel, thte structures were washed in PBS once, then pelleted by gravity. The organoids were dissociated into single cells in TrypLE Express for 10-12 minutes at 37 degrees. The suspensions were washed once PBS and filtered through a 70 um reversible cell strainer. The cells were then pelleted by centrifugation at 400 xg for 4 minutes, resuspended and labelled using BD Human Single Cell Multiplexing Kit. Labelled samples, day 7 (SampleTag_02) and day 18 (SampleTag_06), were pooled into a single cell suspension. Cells were loaded into BD Rhapsody cartridge and libraries were prepared using the BD Whole Transcriptome Amplification (WTA) kit at the Single Cell Sequencing core at Harvard Medical School. The sample library was combined with the Sample Tag library (3%), and sequencing using an Illumina NextSeq 550 High Output kit at the Biopolymers Facility at Harvard Medical School.