Feb 08, 2026
RNA Extraction, Library Preparation, Bulk RNA Sequencing, and Analysis of Mouse Proximal Large Intestine
- Anastasiya Moiseyenko1
- 1California Institute of Technology
- Faecalibacterium prausnitzii improves motor deficits in a-synuclein overexpressing mice
Protocol Citation: Anastasiya Moiseyenko 2026. RNA Extraction, Library Preparation, Bulk RNA Sequencing, and Analysis of Mouse Proximal Large Intestine. protocols.io https://dx.doi.org/10.17504/protocols.io.6qpvry7wogmk/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: February 08, 2026
Last Modified: February 10, 2026
Protocol Integer ID: 242882
Keywords: RNA extraction, library preparation, bulk RNA sequencing, RNA analysis, differentially expressed genes, proximal large intestine, mouse, large intestine this protocol detail, procedure for the rna extraction, proximal large intestine, bulk rna sequencing, rna extraction, large intestine, bulk rna, genes in the mouse, analysis of mouse proximal, expressed gene, mouse proximal
Funders Acknowledgements:
Aligning Science Across Parkinson’s
Grant ID: Grant ID: ASAP-020495
Aligning Science Across Parkinson’s
Grant ID: Grant ID: ASAP-000375
Heritage Medical Research Institute
Grant ID: n/a
Abstract
This protocol details the procedure for the RNA extraction, library preparation, bulk RNA sequencing, and analysis used to assess differentially expressed genes in the mouse proximal large intestine.
Materials
- TRIzol Reagent (Zymo Research)
- Direct-zol RNA MiniPrep Plus Kit (Zymo Research)
- DNase I (included with kit)
- NanoDrop spectrophotometer
- Agilent 2100 Bioanalyzer (Agilent Technologies)
- ABclonal FAST RNA-seq Library Prep Kit
- Illumina NovaSeq X sequencing platform
- STAR aligner v2.7.1
- Mus musculus GRCm36 Primary Assembly reference genome (GENCODE Release M36)
- featureCounts
- R v4.3.0
- DESeq2 R package
- clusterProfiler R package
Troubleshooting
Tissue collection
Dissect approximately 1 cm of proximal large intestine from each mouse.
Immediately place tissue into TRIzol reagent (Zymo Research) and flash-freeze on dry ice.
Store samples at –80 °C until RNA extraction.
Total RNA Extraction
Thaw samples on ice.
Extract total RNA using the Direct-zol RNA MiniPrep Plus Kit (Zymo Research) according to the manufacturer’s protocol.
Perform on-column DNase I treatment as specified by the kit to remove genomic DNA.
Measure RNA concentration and purity using a NanoDrop spectrophotometer.
Assess RNA integrity using an Agilent 2100 Bioanalyzer.
Library Preparation and Sequencing
Prepare RNA-seq libraries using the ABclonal FAST RNA-seq Library Prep Kit according to the manufacturer’s instructions.
Perform sequencing using 50 bp paired-end reads on an Illumina NovaSeq X platform with a target a sequencing depth of ~30 million reads per sample.
Bioinformatics Analysis
Align raw sequencing reads to the Mus musculus GRCm36 Primary Assembly reference genome (GENCODE Release M36) using STAR aligner v2.7.1 with default parameters.
Quantify gene-level read counts using featureCounts. Retain only protein-coding genes with a minimum total count ≥ 10 across all samples.
Import count data into R (v4.3.0). Normalize counts and perform differential expression analysis using the DESeq2 package.
Identify differentially expressed genes by an adjusted p-value < 0.05 and Log2 fold change threshold as defined per comparison.
Perform gene ontology and pathway enrichment analyses using the clusterProfiler package. Using the following criteria: Adjusted p-value < 0.1, log2 fold change > 0.2