UMB Data Catalog

A project of the Health Sciences and Human Services Library
UMB Dataset

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

UID: 209
Author(s): Thomas V. Rousselle, Jennifer M. McDaniels, Amol C. Shetty, Elissa Bardhi, Daniel G. Maluf...
* Corresponding Author
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
Subject Domain
High-Throughput Nucleotide Sequencing
Keywords
RNA-Seq

Access

Restrictions
Unrestricted access
Instructions
The raw sequence data generated in this study were deposited in the GEO
Access via GEO

Raw sequence data
Accession #: GSE195719

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