GSM4509002
GSM4509002: Ang; Homo sapiens; RNA-Seq
Kidney organoids were dissociated to single cells using TrypLE Select (Thermo Fisher Scientific) at 37°C with mechanical trituration. Single cell suspensions were visually inspected under a microscope, counted using an automated hand-held cell counter (Scepter™ 2.0 Cell Counter), and resuspended at a concentration of 15,000 single-cells in 50 microliters of FACS buffer (0.1% BSA–PBS). All four samples were processed according to 10X Genomics ChromiumTM Next GEM Single Cell 3' Reagent Kits v3.1 as per manufacturer's protocol.  Libraries were prepared according to 10X Genomics ChromiumTM Single Cell 3' Reagent Guidelines v3.1 Chemistry as per the manufacturer's protocol. Briefly, single cells were partitioned together with barcoded Gel Bead-In-EMulsions (GEMs) using 10x GemCodeTM Technology. This process lysed cells and enabled barcoded reverse transcription of RNA, generating cDNA from poly-adenylated mRNA. DynaBeads® MyOneTM Silane magnetic beads were used to remove leftover biochemical reagents, then cDNA was amplified by PCR. Quality control size gating was used to select cDNA amplicon size prior to library construction. Read 1 primer sequences were added to cDNA during GEM incubation. P5 primers, P7 primers, i7 sample index, and Read 2 primer sequences were added during library construction. Quality control and cDNA quantification was performed using Agilent D1000 ScreenTape System. Libraries were pooled and first sequenced on NovaSeq 100 (plus 25 additional) Cycle SP dual lane flow cell to approximate the number of recovered cells in each sample. We recovered 4,235 cells in Control, 5,702 cells in Losartan-alone, 5,800 cells in Angiotensin II-alone, and 5,855 cells in Angiotensin II plus Losartan treated groups with an estimated doublet rate of ≈4%. Based on these, we determined proportions for Illumina NovaSeq 100 (plus 25 additional) Cycle S2 dual lane flow cell with a targeted sequencing depth of ~30,000 reads per cell. Illumina sequencer's BCL files were demultiplexed using cellranger mkfastq (a wrapper around Illumina's bcl2fastq). Downstream barcode processing, alignment to pre-built GRCh38, and single-cell 3′ gene counting were performed using CellRanger software version 3.0.1 (10X Genomics). All four libraries were aggregated using cellranger aggr (with mapped normalization). The resulting filtered feature-barcode matrix was imported into Seurat v3 for quality control, dimensionality reduction, cell clustering, and differential expression analysis using default or recommended parameters. 
GEO
GSM4509002
GEO Accession
GSM4509002