Summary: We report the early transcriptional changes in human diabetic nephropathy by single nucleus RNA sequencing
Overall Design: Single nucleus RNA sequencing of 3 early human diabetic kidney samples and 3 controls. Samples represent non-tumor tissue in patients undergoing nephrectomy for renal mass.
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Growth Protocol: | ES cell–derived NS cells were routinely generated by re-plating d 7 adherent neural differentiation cultures (typically 2–3 × 10e6 cells into a T75 flask) on uncoated plastic in NS-A medium (Euroclone, Milan, Italy) supplemented with modified N2 and 10 ng/ml of both EGF and FGF-2 (NS expansion medium). |
Treatment Protocol: | - |
Extract Protocol: | Nuclei were isolated with Nuclei EZ Lysis buffer (NUC-101; Sigma-Aldrich) supplemented with protease inhibitor (5892791001; Roche) and RNase inhibitor (N2615; Promega and AM2696; Life Technologies). Samples were cut into <2-mm pieces and homogenized using a Dounce homogenizer (885302-002; Kimble Chase) in 2 ml of ice-cold Nuclei EZ Lysis buffer, and they were incubated on ice for 5 minutes with an additional 2 ml of lysis buffer. The homogenate was filtered through a 40-μm cell strainer (43-0040-1; pluriSelect) and then centrifuged at 500xg for 5 minutes at 4℃. The pellet was resuspended and washed with 4 ml of the buffer, and then, it was incubated on ice for 5 minutes. After another centrifugation, the pellet was resuspended in Nuclei Suspension Buffer (1xPBS, 0.07% BSA, and 0.1% RNase inhibitor), filtered through a 20-μm cell strainer (43-0020-50; pluriSelect), and counted. |
Library Construction Protocol: | Libraries were prepared according to Illumina's instructions and accompanying 10X Genomics Chromium Single Cell 5' Library and Gel Bead Kit |
Molecule Type: | poly(A)+ RNA |
Library Source: | |
Library Layout: | PAIRED |
Library Strand: | Forward |
Platform: | ILLUMINA |
Instrument Model: | Illumina NovaSeq 6000 |
Strand-Specific: | Specific |
Data Resource | GEN Sample ID | GEN Dataset ID | Project ID | BioProject ID | Sample ID | Sample Name | BioSample ID | Sample Accession | Experiment Accession | Release Date | Submission Date | Update Date | Species | Race | Ethnicity | Age | Age Unit | Gender | Source Name | Tissue | Cell Type | Cell Subtype | Cell Line | Disease | Disease State | Development Stage | Mutation | Phenotype | Case Detail | Control Detail | Growth Protocol | Treatment Protocol | Extract Protocol | Library Construction Protocol | Molecule Type | Library Layout | Strand-Specific | Library Strand | Spike-In | Strategy | Platform | Instrument Model | Cell Number | Reads Number | Gbases | AvgSpotLen1 | AvgSpotLen2 | Uniq Mapping Rate | Multiple Mapping Rate | Coverage Rate |
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