Jan 12, 2026

Public workspaceBulk Astrocyte RNA Seq Extraction

DOI
https://dx.doi.org/10.17504/protocols.io.q26g77y29gwz/v1
  • Mackenzie Ferguson1
  • 1Drexel University College of Medicine
  • MF Space
Mackenzie Ferguson
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DOI: https://dx.doi.org/10.17504/protocols.io.q26g77y29gwz/v1
Protocol Citation: Mackenzie Ferguson 2026. Bulk Astrocyte RNA Seq Extraction. protocols.io https://dx.doi.org/10.17504/protocols.io.q26g77y29gwz/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: In development
We are still developing and optimizing this protocol
Created: January 10, 2026
Last Modified: January 12, 2026
Protocol Integer ID: 238407
Keywords: seq, rna, extraction, bulk
Disclaimer
Only draft form
Abstract
Bulk Astrocyte RNA Seq preparation
Troubleshooting
Cell Preparation
Assume microglia and OPCs have been removed prior to this procedure, if not refer to proper protocols to have pure astrocyte culture
Remove media and discard

Rinse twice with PBS, aspirate the PBS, add 5 ml trypsin-EDTA (.05%) and incubate in the CO2 incubator at 37 °C. Check detachment of astrocytes every 5 min and enforce detachment of astrocytes by hitting the flask against the palm of your hand (2-3 times)
After astrocytes are detached from the culture flask, add 5 ml of astrocyte culture media to inactivate trypsin
Cell Counting
Take a previously prepared 1.5 mL microtube (with Amount10 µL of trypan blue) and combine with Amount10 µL of cell suspension

Load a hemacytometer with 10 μL of the cells and trypan blue solution and examine immediately under a microscope at low magnification.
Count the number of viable cells. Those will light up white and are not invaded with the blue dye
To calculate the number of viable cells per mL of culture, use the formula: Number of viable cells × 104 = cells/mL culture. Remember to correct for the dilution factor.
For Plasmidsaurus e cell couFor Plasmidsaurus bulk RNA seq, recommended cell count per well is 2*105
Transfer proper amount of cell suspension to microtubes. Spin cells at 180 x g for 5 min, aspirate supernatant and add Amount50 µL of Zymo 1x DNA/RNA shield and resuspend

If the lysed cell solution appears highly viscous, vortex it vigorously for about 30s to shear chromosomal DNA.
Transfer prepared supernatant to properly labeled Amount200 µL strip tubes or 96 well plate according to https://plasmidsaurus.com/sample-prep/rna#section1 instructions