Description: Researchers present an optimized protocol for single nuclei RNA sequencing (snRNA-seq) that is fast, low, cost, and time effective due to the elimination of cell sorting and ultra-centrifugation. Both human and mouse tissue biopsies were assessed. Human kidney graft biopsies were collected from five kidney transplant recipients. Mouse needle biopsies were collected from heart, liver, kidney, spleen, lung, duodenum, large intestine, bone marrow and brain.

Subject of Study

Human

Mouse

Subject Domain

High-Throughput Nucleotide Sequencing

Keywords

RNA-Seq

Access via GEO
Raw sequence data
Accession #: GSE195719
Access Restrictions: Unrestricted access
Access Instructions: The raw sequence data generated in this study were deposited in the GEO
Associated Publications: Rousselle TV, McDaniels JM, Shetty AC, Bardhi E, Maluf DG, Mas VR. An optimized protocol for single nuclei isolation from clinical biopsies for RNA-seq. Sci Rep. 0222 June 14; 12:9851.
Data Type: Digital Images

Genomic DNA
Equipment Used: ActinGreen
A selective, high-affinity F-actin probe (phalloidin) conjugated to our bright, photostable, green-fluorescent Alexa Fluor 488 dye.

BioAnalyzer 2100
Automated electrophoresis tool for the sample quality control of biomolecules

Chromium Next GEM Single Cell 5ʹ Kit v2
Analyzer used for high throughput full range single cell analysis. Chromium X runs high-throughput assays. It runs all single cell assays, including high-throughput assays for Single Cell Immune Profiling and Single Cell Gene Expression

Countess II FL Automated Cell Counter
Benchtop automated cell counter. It can perform cell counts as well as viability and cell calculations.

DAPI
Nuclear and chromosome counterstain that emits blue fluorescence upon binding to AT regions of DNA.

Flowmi Cell Strainer
Fast and efficient filtering of small volume samples

Illumina NovaSeq 6000 Sequencing System
Performs whole-genome sequencing

Mitotracker CMXros
Cell-permeant probes stain active mitochondria in live cells for labeling and localization in fluorescent cell imaging.

Sorvall Legend Micro 21
Ventilated centrifuge that offers productivity, versatility, safety and convenience in a compact, easy-to-use lab instrument

Sorvall ST 16
Centrifuge that can perform cell culture and blood processing applications

ThermoFisher Qubit fluorometer
Benchtop fluorometer designed to accurately measure DNA, RNA, and protein quantity. Measures RNA integrity and quality.
Software Used: 10x Genomics Cellranger DNA
Software analysis pipelines that process Chromium single cell DNA sequencing output to align reads, identify copy number variation, and compare heterogeneity among cells. Used in processing of single cell DNA sequencing performed on 10x Chromium platform.

Seurat
Software as R package designed for QC, analysis, and exploration of single cell RNA-seq data. Enable users to identify and interpret sources of heterogeneity from single cell transcriptomic measurements, and to integrate diverse types of single cell data.
Study Type: Interventional
Dataset Format(s): SOFT, TXT, MINiML, TAR
Data Collection Instruments: Principle component analysis

Uniform manifold approximation and projection
Dataset Size: 167 Mb

Grant Support: R01DK109581/National Institute of Diabetes and Digestive and Kidney Diseases (U.S.)

R01DK122682/National Institute of Immunology, Allergy and Infectious Diseases

1U01AI170050/National Institute of Immunology, Allergy and Infectious Diseases

UMB Data Catalog

An optimized protocol for single nuclei isolation from clinical biopsies for RNA-seq
UMB Dataset

An optimized protocol for single nuclei isolation from clinical biopsies for RNA-seq UMB Dataset

An optimized protocol for single nuclei isolation from clinical biopsies for RNA-seq
UMB Dataset