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Sample GSM8015446

Query DataSets for GSM8015446

Status

Public on Jan 16, 2024

Title

Sorted renal cells, KO, Early, Male, tdTomato Negative, Rep 3

Sample type

SRA

Source name

Kidney

Organism

Mus musculus

Characteristics

tissue: Kidney
cells: FAC-sorted cells
facs tdtomato_status: Negative
mouse genotype: Vhl(KO/flox) (KO)
cellular vhl_status: Vhl(KO/wt)
timepoint: Early (1-3 weeks)

Treatment protocol

Five consecutive daily doses of 2 mg tamoxifen were administered to Control and KO mice >20 g by oral gavage using sterile, disposable plastic feeding tubes (Instech FTP-20-38).

Growth protocol

All experimental procedures were conducted in line with AACR guidelines following approval by the Medical Science Ethical Review Committee of the University of Oxford and authorization under UK Home Office regulations of Animals (Scientific Procedures) Act 1986. Mice were housed in individually ventilated cages on a 13 h light/ 11 h dark cycle with food and water provided ad libitum.

Extracted molecule

polyA RNA

Extraction protocol

Kidneys were dissociated using the Multi-Tissue Dissociation Kit 2 (Miltenyi 130-110-203). Briefly, the renal capsule was removed, and the tissue macerated for 5 min on a bed of ice. The macerated kidney was then transferred to 1.45 ml Buffer X, 30µl of Enzyme D, 15 µl of Enzyme P, 15 μl of Buffer Y, and 6 µl of Enzyme A, all prepared according to the manufacturer’s instructions. The tube was then agitated under water at 37˚C in a shaking incubator at 150 rpm for 30 min. Dissociation was stopped by the addition of 150 µl Fetal Bovine Serum (FBS; Sigma F7524), and resuspension in 9 ml of RPMI-1460 medium (Merck R0883). Undigested tissue was removed using a 40 µm cell strainer and the filtrate centrifuged (300g for 10 min at 4˚C). Erythrocytes were eliminated by resuspending dissociated cells in 3 ml of 1x RBC Lysis Buffer (Miltenyi 130-094-183) prepared in deionized water for 2 min at RT. Cells were centrifuged (300g for 5 min at 4˚C) and resuspended in ice-cold D-PBS before being counted on a Thermo Scientific Countess II machine for total yield and viability. Dissociated cells were resuspended in 10% FBS 2 mM EDTA in D-PBS (Thermo 14190144) to a concentration of 5 million live cells per ml. 10 μl of 10 μg/ml DAPI (Sigma D9542) was added to stain for viability. Cells were sorted using a BD Aria Fusion Cell Sorter. tdTomato was excited with a 561 nm laser and fluorescence detected with a 582/15 band pass filter. DAPI was excited with a 405 nm laser and fluorescence detected with a 450/40 band pass filter. Live, single, tdTomato-positive and -negative cells were collected in FBS-coated polypropylene tubes and pelleted by centrifugation at 300g for 10 min. Cells were counted again for yield and viability and processed for single-cell sequencing.
Sorted cells were prepared into single-cell droplets using the Chromium Next GEM Single-cell kit. 20,000 live cells were loaded per sample on separate Chromium Next GEM Chip G (10X Genomics PN-1000127) channels. cDNA clean-up, amplification and adaptor ligation were performed with the Chromium Next GEM Single Cell 3ʹ Kit v3.1 (10X Genomics PN-1000268). Twelve reaction cycles were used for cDNA amplification. cDNA yield was quantified using the High Sensitivity D1000 ScreenTape Assay (Agilent 5067-5584 and 5067-5585) to optimize the number of reaction cycles for library preparation. Single cell sequencing libraries were prepared using Dual Index Kit TT Set A (10X Genomics PN-1000215) sequence indices. Prepared libraries were quantified with KAPA Library Quantification Kit (Roche KK4824) following the manufacturer’s protocol.

Library strategy

RNA-Seq

Library source

transcriptomic single cell

Library selection

cDNA

Instrument model

NextSeq 2000

Description

10x Genomics
OY1166_neg

Data processing

Single-cell libraries were sequenced on Illumina NextSeq 2000 sequencing system with P3 200 cycle (Illumina 20040560) reagents. Equimolar pools of up to six sample libraries were prepared and diluted to 650 pM and mixed with 1% PhiX Control v3 DNA (Illumina FC-110-3001) in RSB buffer to a final volume of 25 μl. Sequencing was performed and preliminary sequencing results (bcl files) and FASTQ files were generated with the DRAGEN FASTQ Generation 3.8.4 or the DRAGEN BCL convert workflow optimized for Single Cell RNA Library Kit 1 library prep kit, and the Single Cell RNA Index Adaptors 1-B index adaptor kit, with 28 and 152 Read 1 and 2 cycles respectively
The mm10 reference genome was customized and the starting reference fastq files and GTF files came from refdata-gex-mm10-2020-A.tar.gz and are available for download at https://support.10xgenomics.com/single-cell-gene-expression/software/downloads or https://cf.10xgenomics.com/supp/cell-exp/refdata-gex-mm10-2020-A.tar.gz. The transcript sequence for Vhlpjr.KO that included the tdTomato sequence was then added manually to the fasta reference genome file and annotation for tdTomato transcript was added manually to the gtf file. The cellranger 6.0.1 mkref function was used to build the custom reference genome and reads taken from FASTQ files generated for each sample were aligned to the custom reference genome by CellRanger (version 6.1.1; https://support.10xgenomics.com/single-cell-gene-expression/ software/pipelines/latest/what-is-cell-ranger) using default parameter sample by sample. After aligning, for each read pair, cell barcodes and unique molecular identifiers (UMIs) were obtained from Read 1 and read counts per feature were obtained from Read 2. Only those UMIs that could be linked to a valid cell barcode and a gene exon region were included to create the cell by gene count matrix. Reads from Vhl exon 1 were excluded from analysis to prevent ambiguous alignment. Cells were subjected to the following filters: detected genes > 200, fraction of mitochondrially-encoded reads < 0.5, and detected genes < 3 x median for each sample. The threshold for mitochondrially encoded reads was set to this value in line with published kidney scRNA-seq studies to account for the high mitochondrial content in RTE cells19. Information and quality control metrics for all sequenced samples are provided in Supplementary Table S1. Cells from different samples and sequencing runs were then aggregated, which sometimes required manual renaming of duplicate cell ‘names’. Downstream analysis was conducted using the R package Seurat 4.0.3: Read counts were log-normalized and scaled, PCA was run using the scaled data and UMAP was generated using the top 30 PCs.
Assembly: mm10
Supplementary files format and content: Seurat objects containing information from all the samples and positive samples were provided. They contain the information for all the cells after quality control, for example, raw counts and normalised counts for all the features, dimension reduction information and all the meta information.

Submission date

Jan 12, 2024

Last update date

Jun 21, 2024

Contact name

David Robert Mole

E-mail(s)

david.mole@ndm.ox.ac.uk

Phone

0044 (0)1865 613956

Organization name

University of Oxford