Apr 14, 2026
Bulk RNA-seq Protocol for Astrocytes
- 1Icahn School of Medicine at Mount Sinai
Protocol Citation: Elena Coccia 2026. Bulk RNA-seq Protocol for Astrocytes. protocols.io https://dx.doi.org/10.17504/protocols.io.81wgbj8eyvpk/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: April 14, 2026
Last Modified: April 14, 2026
Protocol Integer ID: 314957
Keywords: RNA extraction, astrocytes, bulk RNA sequencing, gene expression, seq protocol for astrocyte, total rna from astrocyte, astrocyte, bulk rna, subsequent bulk rna, total rna, gene expression profile, rna, analysis of gene expression profile, seq protocol, seq
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Abstract
This protocol aims to extract total RNA from astrocytes for subsequent bulk RNA sequencing, enabling the analysis of gene expression profiles.
Materials
TrypLE Express (Enzyme for cell dissociation) - 10 mL
PBS (Phosphate Buffered Saline) (Ice-cold solution for cell collection) - 50 mL
RLT Lysis Buffer (Qiagen RNeasy kit component) - 1 mL
Qiagen RNeasy Kit (RNA extraction kit) - 1 kit
Liquid Nitrogen (For cell freezing) - As needed
Reagents:
Enzyme TrypLE Express - A recombinant enzyme for cell dissociation
Buffer RLT Lysis Buffer - Buffer for lysing cells to extract RNA
Troubleshooting
Problem
Low RNA yield
Solution
Ensure proper cell scraping and lysis. Verify enzyme activity.
Problem
RNA degradation
Solution
Keep samples on ice and work quickly. Use RNase-free reagents.
Safety warnings
Handle all reagents and samples with care to avoid exposure to harmful substances. Use appropriate personal protective equipment (PPE) including gloves and lab coats.
Cell Collection
Remove media from 2D astrocyte cultures.
Cell Collection
2. Scrape cells directly into ice-cold PBS (50 mL).
Centrifuge the scraped cells at 1,000 × g for 5 minutes at 4 °C.
Discard the supernatant.
RNA Extraction
Extract total RNA using the Qiagen RNeasy kit.
Lysis and Binding
Add 300µL of RLT lysis buffer to the pelleted cells
Proceed with the Qiagen RNeasy kit protocol for RNA following the manufacturer's instructions
RNA Quantification
Quantify RNA using a Nanodrop or similar device and assess RNA quality
Sequencing Submission
Submit 1 µg of total RNA to Azenta/GENEWIZ for library preparation and sequencing.
Library Preparation (performed by GENEWIZ)Untitled section
polyA selection for mRNA enrichment
Standard Illumina-compatible library preparation, single indexing
Sequencing (performed by GENEWIZ)Untitled section
Illlumina HiSeq platform. Paired-end, 150 bp reads
Data Processing
Pseudo-align FASTQ files to reference genome GRCh38.p13 using kallisto
Differential expression analysis with edgeR in R (v2023.12.0+369)
Visualization and functional enrichment with ggplot2 and clusterProfiler
Protocol references