Oct 29, 2025

Public workspaceRNA Sequencing

DOI
https://dx.doi.org/10.17504/protocols.io.e6nvw42k7lmk/v1
  • Dahlia Rohm1,2,
  • Joshua Black1,2,
  • Sean R. McCutcheon1,2,
  • Alejandro Barrera1,2,
  • Shante Berry1,2,
  • Daniel Morone1,2,
  • Xander Nuttle3,4,
  • Celine de Esch3,4,
  • Derek Tai3,4,
  • Michael Talkowski3,4,
  • Nahid Iglesias1,
  • Charles A. Gersbach1,2
  • 1Duke University;
  • 2Center for Advanced Genomic Technologies;
  • 3Massachusetts General Hospital;
  • 4Broad Institute of Massachusetts Institute of Technology and Harvard University
  • Gersbach Lab
Andrea R Daniel
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DOI: https://dx.doi.org/10.17504/protocols.io.e6nvw42k7lmk/v1
Protocol Citation: Dahlia Rohm, Joshua Black, Sean R. McCutcheon, Alejandro Barrera, Shante Berry, Daniel Morone, Xander Nuttle, Celine de Esch, Derek Tai, Michael Talkowski, Nahid Iglesias, Charles A. Gersbach 2025. RNA Sequencing. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvw42k7lmk/v1
Manuscript citation:
Activation of the imprinted Prader-Willi Syndrome locus by CRISPR-based epigenome editing.
Rohm D, Black JB, McCutcheon SR, Barrera A, Berry SS, Morone DJ, Nuttle X, de Esch CE, Tai DJC, Talkowski ME, Iglesias N, Gersbach CA. Cell Genomics. 2025 Feb 12;5(2):100770. doi: 10.1016/j.xgen.2025.100770. PMID: 39947136
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: October 08, 2025
Last Modified: October 29, 2025
Protocol Integer ID: 229362
Keywords: RNA Sequencing, iPSC, Prader-Willi Syndrome, induced pluripotent stem cell, pluripotent stem cell, methods for rna, rna
Funders Acknowledgements:
NIH
Grant ID: HG012053
Disclaimer
This protocol was adapted from the work of Dahlia Rohm and colleagues in the Gersbach lab at Duke University.
Abstract
This protocols describes methods for RNA sequencing in human induced pluripotent stem cells.
Materials
Norgen Total RNA Purification Plus Micro kit (Norgen, 48500); Qubit RNA Broad Range quantification assay (Thermo, Q10211); Agilent TapeStation (Agilent TapeStation 4200) RNA ScreenTape (Agilent, 5067-5576); Genewiz (Azenta Life Sciences); Trimmomatic v0.32; STAR aligner; featureCounts; DESeq2.
Troubleshooting
Cell preparation and RNA preparation
Harvest cells, protocol defined by user. Pellet ~100,000 cells (between 1000 and 500,000) and proceed to RNA isolation immediately or flash-freeze in liquid nitrogen and store pellets at -80°C until processing.
Isolate total RNA using Norgen Total RNA Purification Plus Micro kit (Norgen, 48500).
RNA sequencing
Quantify RNA with the Qubit RNA Broad Range quantification assay (Thermo, Q10211) and optionally validate RNA integrity with Agilent TapeStation (Agilent TapeStation 4200) RNA ScreenTape (Agilent, 5067-5576) or a similar automated electrophoresis system.
Submit total RNA to Genewiz (Azenta Life Sciences) for total RNA sequencing for 2x150bp paired-end short read sequencing. Genewiz verifies quality of samples and libraries and performed DNAse treatment as necessary.
Subject resulting reads to adapter trimming using Trimmomatic v0.32, align to GRCh37 or GRCh38 with the STAR aligner, and retrieve gene counts using featureCounts.
Perform differential expression analysis using DESeq2. Determine genes with significant differential expression using a threshold of FDR<0.01 and absolute value of Log2(FC)>1.