Nov 30, 2023
RNAseq of primary human T cells overexpressing BATF3
- Sean R. McCutcheon1,
- Adam M. Swartz1,
- Michael C. Brown1,
- Alejandro Barrera1,
- Christian McRoberts Amador1,
- Keith Siklenka1,
- Lucas Humayun1,
- Maria A. ter Weele1,
- James M. Isaacs1,
- Andrea Daniel1,
- Timothy E. Reddy1,
- Andrew Allen1,
- Smita K. Nair1,
- Scott J. Antonia1,
- Charles A. Gersbach1
- 1Duke University
- Andrea Daniel: This protocol was adapted from Sean McCucheon's work in the Gersbach lab at Duke University.
- Gersbach Lab
Protocol Citation: Sean R. McCutcheon, Adam M. Swartz, Michael C. Brown, Alejandro Barrera, Christian McRoberts Amador, Keith Siklenka, Lucas Humayun, Maria A. ter Weele, James M. Isaacs, Andrea Daniel, Timothy E. Reddy, Andrew Allen, Smita K. Nair, Scott J. Antonia, Charles A. Gersbach 2023. RNAseq of primary human T cells overexpressing BATF3. protocols.io https://dx.doi.org/10.17504/protocols.io.3byl4q1ejvo5/v1
Manuscript citation:
McCutcheon, S.R., Swartz, A.M., Brown, M.C. et al. Transcriptional and epigenetic regulators of human CD8+ T cell function identified through orthogonal CRISPR screens.Nat Genet (2023). https://doi.org/10.1038/s41588-023-01554-0
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: November 30, 2023
Last Modified: November 30, 2023
Protocol Integer ID: 91589
Keywords: RNAseq, CD8+ T cells, BATF3, performing rnaseq, rnaseq, overexpressing batf3, using human cd8, human cd8, cell
Funders Acknowledgements:
NIH
Grant ID: HG012053
Abstract
This is a protocol describes methods for performing RNAseq using human CD8+ T cells overexpressing BATF3 or a GFP control.
Transfections for high-titer lentiviral production
Plate 1.2 x 106 or 7 x 106 HEK293T cells in a 6 well plate or 10 cm dish in the afternoon with 2 mL or 12 mL of complete opti-MEM (Opti-MEM‱ I Reduced Serum Medium supplemented with 1x Glutamax, 5% FBS, 1 mM Sodium Pyruvate, and 1x MEM Non-Essential Amino Acids).
The next morning, transfect HEK293T cells with 0.5 μg pMD2.G, 1.5 μg psPAX2, and 0.5 μg transgene for 6 well plates or 3.25 μg pMD2.G, 9.75 μg psPAX2, and 4.3 μg transgene for 10 cm dishes using Lipofectamine 3000.
Exchanged media 6 hours after transfection and collect and pool lentiviral supernatant at 24 hours and 48 hours after transfection.
Primary human CD8+ T cell cultures
Isolated CD8+ T cells from individual donors were obtianed directly from vials purchased from StemCell Technologies.
Culture T cells were in PRIME-XV T cell Expansion XSFM (FujiFilm) supplemented with 5% human platelet lysate (Compass Biomed), 100 U ml−1 penicillin and 100 μg ml−1 streptomycin. All media were supplemented with 100 U ml−1 human IL-2 (Peprotech).
Transduction of primary human CD8+ T cells
Centrifuged lentiviral supernatant at 600g for 10 min to remove cellular debris.
Concentrate lentivirus to 50–100× the initial concentration using Lenti-X Concentrator (Takara Bio).
Transduce T cells at 5–10% v/v of concentrated lentivirus at 24 h post-activation. For dual transduction experiments, T cells were serially transduced at 24 h and 48 h.
RNAseq
T cells overexpressing either BATF3 or GFP were cultured for 10 days post transduction.
RNA was isolated using Norgen’s Total RNA Purification Plus Kit and submitted to Azenta (formerly Genewiz) for standard RNA-seq with polyA selection.
Reads were first trimmed using Trimmomatic72 v0.32 to remove adapters and then aligned to GRCh38 using STAR v2.4.1a aligner.
Gene counts were obtained with featureCounts73 from the subread package (version 1.4.6-p4) using the comprehensive gene annotation in Gencode v22.
Differential expression analysis was determined with DESeq2 (ref. 68) where gene counts are fitted into a negative binomial generalized linear model and a Wald test determines significant DEGs.
Upregulated and downregulated DEGs were input into EnrichR’s GO Biological Processes 2021 database71 for functional annotation.