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| Status |
Public on Feb 26, 2024 |
| Title |
tdTnegGFPpos cells at d6 post-IRI, Tamoxifen at d2 and d4 post-IRI, FACS-enriched, biol rep 3 |
| Sample type |
SRA |
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| Source name |
Kidney-3
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| Organism |
Mus musculus |
| Characteristics |
tissue: Kidney-3
cell type: endogenous GFP cells only from kidney-3
genotype: Axin2CreERT2/+:R26RtdT/+ :Acta2-GFP
age: 9-12 weeks
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| Treatment protocol |
Age- (9 – 12 weeks old) and weight –matched (25 – 30 gm) male mice were subjected to bilateral renal ischemia reperfusion injury (IRI) surgery. To obtain WRCs, tamoxifen was administered at d2, and d4 to Axin2CreERT2/+:R26RtdT/+:Acta2-GFP animals, and at d6 dual positive (tdT positive and GFP positive) and their tdT negative, GFP positive counterparts were enriched via FACS. The cells were isolated as stated in the below stated extract protocol.
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| Growth protocol |
N/A
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
Briefly cortices and outer medulla regions of the kidney were dissected and thoroughly minced in 1mlcold DMEM/F12 media using sterile fine forceps. The minced tissue resuspended in fresh 1ml DMEM/F12 media supplemented with 7.5mg/ml of B. Licheniformis Cold Active Protease, 125U/ml DNase1 and 0.1mg/ml Liberase TL and continuously kept on ice for 1 hour with frequent pipetting using 1ml pipette every 10 min. After complete dissociation of tissues into single cells (an aliquot of cell suspension was taken every 20 min to check dissociation of the tissue under the microscope), the enzymatic reaction was stopped using cold DMEMF12 media + 10% FBS. The cell suspension was filtered through a 40μm cell strainer. The filtrate was then centrifuged at 300g for 5 min and transferred to FACS tubes (BD Biosciences). Cells were washed in PBS and resuspended in 1.5ml of FACS sorting buffer containing 1mg/ml DAPI (1:1000, Life Technologies). Cell sorting was performed using the FACSAria III cell sorter (BD Biosciences). Cell populations were gated to exclude debris and doublets using FACSDiva software (BD Biosciences). Cells were collected from relevant gated cell populations in 1.5mL collection tubes (Eppendorf) containing collection buffer. RNA was extracted from the above desired cells following the manufacture instruction (mRNA easy, Qiagen). Briefly, total RNA samples were assessed for concentration using a Nanodrop (Nanodrop 504 ND8000, Thermo Fisher Scientific, Carlsbad, CA) and quality using the TapeStation 505 (4200 TapeStation, Agilent Technologies, Santa Clara, CA). Up to 30 ng of total RNA per sample was used for poly-A mRNA selection.
Libraries for RNA-Seq were prepared with Nugen Universal plus mRNA-Seq Kit (part number: 0508) to generate strand-specific RNA-seq libraries. The workflow consists of poly(A) RNA selection, RNA fragmentation and double-stranded cDNA generation using a mixture of random and oligo(dT) priming, followed by end repair to generate blunt ends, adaptor ligation, strand selection, and PCR amplification to produce the final library. Different index adaptors were used for multiplexing samples in one sequencing lane. The concentration of the amplified library was measured with a Qubit fluorometer and an aliquot of the library was resolved on a Bioanalyzer.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
B6
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| Data processing |
Raw sequencing data was demultiplexed and converted to fastq format by using bcl2fastq v2.20(Illumina, San Diego, California). Then reads were aligned to the transcriptome using STAR(version 2.6.1) (Dobin A et al., 2013) / RSEM (version 1.2.28) (Li B and Dewey CN, 2011) withdefault parameters, using a custom MOUSE GRCh38 transcriptome reference downloaded fromhttp://www.gencodegenes.org, containing all protein coding and long non-coding RNA genesbased on MOUSE GENCODE version 33 annotation. Expression counts for each gene in allsamples were normalized by a modified trimmed mean of the M-values normalization method andthe unsupervised PC analysis (PCA) was performed with DESeq2 Bioconductor package version1.26.0 in R version 3.6.3. Each gene was fitted into a negative binomial generalized linear model,and the Wald test was applied to assess the differential expressions between two sample groups byDESeq2. Benjamini and Hochberg procedure was applied to adjust for multiple hypothesis testing,and differential expression gene candidates were selected with a false discovery rate less than 0.05.
Further filtering of candidate differentially expressed genes to false discovery rate less than 0.01and an absolute log 2 fold-change greater than 1 was applied to the studies comparing normaluninjured PTEC vs. Sox9:48h IRI vs. Sox9: day14 post IRI. GO term enrichment was conductedwith DAVID retaining only terms with a p-value for enrichment less than 0.01. After manuallymerging redundant biological process GO terms, relevant terms among the top 20 were plottedusing custom scripts in R and the ggplot2 (version 3.3.3) package. For visualization of coordinatedgene expression in samples, a two-way hierarchical clustering with Pearson correlation distancematrix was performed with samples and DEG candidates using the Bioconductor g-plots package(version 3.0.3) in R. MA plots, and PCA plots were created using custom R scripts and theBioconductor FactoMineR package (version 2.4).
Assembly: mm10
Supplementary files format and content: Tab-delimited text file includes raw counts for each sample
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| Submission date |
Dec 08, 2023 |
| Last update date |
Feb 26, 2024 |
| Contact name |
AGCT Core |
| Organization name |
Cedars Sinai Medical Center
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| Department |
BMS
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| Street address |
8687 Melrose Ave, PDC B230
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| City |
West Hollywood |
| State/province |
CA |
| ZIP/Postal code |
90069 |
| Country |
USA |
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| Platform ID |
GPL24247 |
| Series (2) |
| GSE249777 |
Sox9 switch links regeneration to fibrosis at the single-cell level in mammalian kidneys [RNA-Seq 1] |
| GSE249781 |
Sox9 switch links regeneration to fibrosis at the single-cell level in mammalian kidneys |
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| Relations |
| BioSample |
SAMN38732779 |
| SRA |
SRX22835019 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
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