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Sample GSM6514377

Query DataSets for GSM6514377

Status

Public on Jan 01, 2023

Title

WT1

Sample type

SRA

Source name

kidney

Organism

Mus musculus

Characteristics

tissue: kidney
genotype: WT

Extracted molecule

total RNA

Extraction protocol

To induce transgene expression, VPR;Pax8-rtTa mice were fed DOX grain-based rodent diet at 7 week of age and continued for 11 weeks induction until mice were euthanized. The mice were anesthetized with Ketamine/Xylazine and perfused with chilled 1x PBS via the left ventricle. Kidneys were decapsulated, minced into 1-mm3 pieces with a sterile razor blade, and incubated in 2 mL of digestion buffer containing 1 mg/mL collagenase type II (Sigma), 1 mg/mL Pronase E (Sigma), and 50 mg/mL DNase I (Roche) at 37°C for 15 min. The partially digested tissue was then passed through a 70 mm cell strainer (Falcon) into RPMI medium supplemented with 10% FBS twice on ice and pelleted by centrifugation (200 g for 5 min). Then, the pellet was resuspended in HBSS and sheared with a 27G needle to remove large clumps. They were further filtered through a 40 mm cell strainer (Falcon) to obtain a single-cell solution. After centrifugation, the cell pellets were incubated with 3 mL of RBC lysis buffer (eBioscience 00-4333-57) to remove red blood cells on ice for 5 min. Finally, the cells were washed and resuspended in RPMI medium two times to remove debris or cell aggregates. Cell viability and number were determined using a Countess II Automated Cell Counter (Invitrogen, C10227) after staining with trypan blue. By using this method, we were able to generate single-cell suspensions with >80% viability.
Sequencing libraries were constructed according to 10x standard protocol by following the steps cDNA fragmentation, end repair and A-tailing, size selection by SPRIselect beads (Beckman Coulter), adaptor ligation, sample index PCR amplification, and repeat SPRIselect beads size selection. The final constructed single-cell libraries were sequenced by Illumina Novaseq machine with total reads per cell targeted for a minimum of 50,000.

Library strategy

RNA-Seq

Library source

transcriptomic single cell

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

Description

WT5
10X Genomics

Data processing

The 10X Libraries were sequenced on the Illumina NovaSeq6000 sequencer with pair-end reads (28 bp for read 1 and 91 bp for read 2). The sequencing data were primarily analyzed by the 10X cellranger pipeline (v3.0.2) in two steps. In the first step, cellranger mkfastq demultiplexed samples and generated fastq files; and in the second step, cellranger count aligned fastq files to the 10X pre-built mouse reference (mm10) and extracted gene expression UMI counts matrix.
Genes expressed in less than 3 cells and cells expressing less than 200 genes were considered as low quality and removed from further analysis, and cells expressing more than 5,000 genes were considered as doublets and removed. Cells with more than 20 percent expression from mitochondrial genes were considered to be dead cells and removed.
Assembly: mm10
Supplementary files format and content: RawCounts.txt: Tab-separated values file and matrix file.

Submission date

Aug 29, 2022

Last update date

Jan 01, 2023

Contact name

Zeguo Sun

E-mail(s)

zeguo.sun@mssm.edu

Phone

6466427380

Organization name

Ichan School of Medicine at Mount Sinai