Jan 29, 2026

Public workspaceBulk RNA-seq

DOI
https://dx.doi.org/10.17504/protocols.io.81wgbon1olpk/v1
  • Boxun Li1,
  • Charles Gersbach1
  • 1Duke University
  • Gersbach Lab
Andrea R Daniel
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DOI: https://dx.doi.org/10.17504/protocols.io.81wgbon1olpk/v1
Protocol Citation: Boxun Li, Charles Gersbach 2026. Bulk RNA-seq. protocols.io https://dx.doi.org/10.17504/protocols.io.81wgbon1olpk/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: January 23, 2026
Last Modified: January 29, 2026
Protocol Integer ID: 240233
Keywords: RNA-seq, neurons, astrocytes, performing bulk rna, bulk rna, mouse primary astrocyte, primary astrocyte, seq on ipsc, seq, seq this protocol, derived neuron
Funders Acknowledgements:
NIH
Grant ID: HG012053
Disclaimer
This protocol was adapted from the work of Boxun Li in the Gersbach lab at Duke University.
Abstract
This protocol describes methods for performing bulk RNA-seq on iPSC-derived neurons and mouse primary astrocytes.
Materials
Mr. Frosty Freezing Container (Thermo Scientific, 5100-0001)
dimethyl sulfoxide (Sigma-Aldrich, D8418)
KnockOut Serum Replacement (Gibco, 10828010)
RNeasy Mini Kit (Qiagen 74104)
Qubit RNA Broad Range Assay Kit (Invitrogen Q10211)
RNase‑free DNase Set (Qiagen 79254)
Troubleshooting
Bulk RNA-seq
Collect neuron and mouse primary astrocyte monocultures, as well as neuron-astrocyte co-cultures after 3, 7, 14, and 28 days into neuronal maturation (cell re-seeding on PLO-coated plate and switching to Maturation Medium (as detailed in protocol [Ref 1.] Ngn2 neuron differentiation) (i.e.,  2, 6, 13, and 27 days after co-culturing, respectively). 
  • Note: Astrocyte monoculture is incubated in the identical medium as neuron mono- and co-cultures. 
On the collection days mentioned above, collect the neuron/astrocyte mono- and co-cultures for RNA extraction. Either viably freeze scraped-off cell sheets, dissociated single cells (as detailed in protocol [Ref 2.] neuron dissociation), or directly lyse cells on plate. 
  1. Option 1: To scrape the cells, use a cell scraper to scrape off >90% of the cells. Collect them into a 15mL conical with Neuronal Maturation Medium. Spin at 300xg for 5 mins. Resuspend the pellet in freeze medium (90% KnockOut Serum Replacement (Gibco, 10828010) + 10% dimethyl sulfoxide [Sigma-Aldrich, D8418]). Freeze cells at -80C in Mr. Frosty Freezing Container (Thermo Scientific, 5100-0001). Transfer frozen vials to liquid nitrogen for storage until ready for RNA extraction. 
  2. Option 2: To dissociate the cultures into single cells (refer to protocol [Ref 2.] Neuron dissociation). Store frozen vials in liquid nitrogen until ready for RNA extraction. 
  3. Option 3: Lyse cells on tissue culture plate. Remove medium and add 350µL/well (for 6-well plates) RLT buffer (RNeasy Mini Kit, Qiagen 74104) to lyse the cells. Collect and freeze the lysates at -80C until ready for RNA extraction. 
When all time points are collected, perform RNA extraction following the manufacturer’s recommended protocol for silica‑column purification (RNeasy Mini Kit, Qiagen 74104), with optional incorporation of an on‑column DNase digestion step to remove residual genomic DNA (RNase‑free DNase Set, Qiagen 79254). Elute RNA in small volumes (typically 30µL), optionally as two sequential elutions, in RNase‑free water
  1. For frozen cell sheets or dissociated cells, thaw cells at 37C. Resuspend cells in 5mL Maturation Medium by adding medium dropwise to cells in freeze medium in a conical tube. Spin for 5 mins at 300 xg. Lyse each pellet in 350µL Buffer RLT. 
Measure RNA concentrations using a fluorometric assay (Qubit RNA Broad Range Assay Kit, Invitrogen Q10211) on Invitrogen Qubit 4 Fluorometer. 
Submit RNA samples to Genewiz for RNA-seq library prep (Library preparation, Illumina, RNA with rRNA depletion) and sequencing (Illumina, 2x150bp, ~350M PE reads) services, with targeted sequencing depth for the mono-cultured neurons and astrocytes samples at 30 million paired-end reads per sample, and for the co-cultured neurons and astrocytes samples at 60 million per sample. 
Protocol references
1. Li B, Gersbach CA. Excitatory neuron differentiation from inducible Ngn2 iPSCs (WTC11). https://www.protocols.io/view/excitatory-neuron-differentiation-from-inducible-n-ewov1kqxpgr2/v1


2. Li B, Gersbach CA. Neuron dissociation protocol for single-cell sequencing applications. https://www.protocols.io/view/neuron-dissociation-protocol-for-single-cell-seque-j8nlk1or5g5r/v1