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Sample GSM7361661

Query DataSets for GSM7361661

Status

Public on Oct 13, 2023

Title

Male, Ad lib GEX

Sample type

SRA

Source name

Kidney

Organism

Mus musculus

Characteristics

tissue: Kidney
strain: C57BL/6J
treatment: ad lib food, water deprived

Treatment protocol

mice were either left with ad lib food and water, or the water was removed for 9-11 h and euthanasia and kidney collection occurred at midnight. 1/2 kidney was cut into small 5 mm cubes and snap frozen

Extracted molecule

total RNA

Extraction protocol

Nuclei from that kidney were isolated with Nuclei EZ Lysis buffer (NUC-101; Sigma-Aldrich) supplemented with protease inhibitor (cOmplete™ ULTRA tab; Sigma-Aldrich) . The frozen kidney cubes were placed in a sterile petri dish with 1 ml of Nuclei EZ lysis buffer (plus protease and RNase inhibitors) and quickly minced. The tissue was then homogenized with RNase-free, disposable pellet pestles and tubes (Fisher #12-141-368), and a hand held, Bel-Art™ Micro-Tube homogenizer (Wayne, NJ). The homogenate was filtered through a 40 micron strainer (all strainers were purchase from pluriSelect Life Science). The homogenate was moved to a sterile, RNase-free 15 ml tube with an additional 2 ml of Nuclei EZ lysis buffer (plus protease and RNase inhibitors). After a 5 min incubation on ice, the homogenate was filtered through a 40 micron strainer. The subsequent homogenate was centrifuged at 500 g for 5 min at 4°C. The pellet was resuspending in Nuclei EZ lysis buffer (plus 1 ml/ml of SUPERaseIN and RNAsin), incubated on ice for 5 min and centrifuged again. The pellet was resuspended in 0.5 ml of 10X Genomics 1X nuclei suspension buffer, filtered through a 5 micron strainer, and nuclei immediately counted with a hemocytometer.
5000 nuclei per sample were loaded in the Chromium Next GEM Chip J Single Cell Kit and the DNA libraries generated with the Chromium Next GEM Single Cell Multiome ATAC + Gene Expression Reagent Bundle

Library strategy

RNA-Seq

Library source

transcriptomic single cell

Library selection

cDNA

Instrument model

Illumina NextSeq 500

Description

single nuclues RNA and ATAC-seq, 10X Genomics Multiome
combined.rds
barcodes.xlsx

Data processing

CellRanger arc-2.0.0 GEX and ATAC output files from filtered_peak_bc_matrix. For each sample (630, 636, 641, 638) the metadata is in per_barcode_metrics.csv, ATAC fragments in ATAC_fragments.tsv.gz, GEX counts in features.tsv.gz
These files were then further processed with Seurat v4.3.0 and Signacv1.9. First low quality nuclei were filtered with this criteria sample_630 <- subset( x = sample_630, subset = nCount_RNA > 1000 & nCount_RNA < 25000 & nCount_ATAC > 1000 & nCount_ATAC < 100000 & nucleosome_signal < 2 &TSS.enrichment > 1)
The all peaks were recalled using the package MACS2, and then each sample went through Log Normalization and scale.factor = 10000
Each sample then underwent PCA, JAckStraw, Findneighbors,FindClusters, Run UMAP
All samples were then merged and underwent LogNormalization, ScaleData, RunPCA, JackStraw, FindNeighbors, Findcllusters, RunUMAP
Assembly: mm10
Supplementary files format and content: Seurat Object named: combined.rds
Supplementary files format and content: barcodes.xlsx

Submission date

May 16, 2023

Last update date

Oct 13, 2023

Contact name

Kelly Anne Hyndman

E-mail(s)

hyndmank@uab.edu

Organization name

University of Alabama at Birmingham

Department

Medicine