Jun 02, 2026

Bulk TF ORF screen for regulators of TOX in primary human CD8+ T cells

DOI
https://dx.doi.org/10.17504/protocols.io.5jyl83rk8v2w/v1
  • 1Duke University
  • Gersbach Lab
Andrea R Daniel
Icon indicating open access to content
QR code linking to this content
DOI: https://dx.doi.org/10.17504/protocols.io.5jyl83rk8v2w/v1
Protocol Citation: Christian McRoberts Amador, Charles Gersbach 2026. Bulk TF ORF screen for regulators of TOX in primary human CD8+ T cells. protocols.io https://dx.doi.org/10.17504/protocols.io.5jyl83rk8v2w/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: June 01, 2026
Last Modified: June 02, 2026
Protocol  Integer ID: 318304
Keywords: TOX, T cell exhaustion, mORF TF-wide screen, tox in primary human cd8, tox protein level, regulators of tox, low tox expression for downstream analysis, primary human cd8, cd28 stimulation, bulk tf orf screen for regulator, transcription factor, tox, low tox expression, bulk tf orf screen, tf isoform
Funders Acknowledgements:
NIH
Grant ID: HG012053
Disclaimer
This protocol was adapted from the work of Christian McRoberts Amador and colleagues in the Gersbach lab at Duke University.
Abstract
This protocol describes a transcription factor (TF)–wide gain‑of‑function screening approach to identify regulators of TOX, a key driver of T cell exhaustion. Using pooled overexpression screens, the ability of individual TFs to modulate TOX protein levels across multiple CD8⁺ T cell states will be assessed. Primary CD8⁺ T cells are transduced with the MORF library, which encodes 3,548 TFs and TF isoforms, and cultured either under standard expansion conditions (“expanded”) or under chronic α‑CD3/CD28 stimulation (“exhausted”). Following culture, fluorescence‑activated cell sorting (FACS) is used to isolate populations with high or low TOX expression for downstream analysis.
Materials
mORF pooled lentiviral library, Addgene #192821
FOXP3 TF staining kit, Thermo cat no. 00-5523-00
TOX antibody, Miltenyi Biotec cat no. 130-118-335
PicoPure DNA extraction kit, Applied Biosystems cat no. KIT0103
NEBNEXT 2× Master Mix, NEB cat no. M0541L
Ampure XP beads for PCR Cleanup, Beckman Coulter cat no. A63882
High Sensitivity D1000 Reagents, Agilent Technologies cat no. 5067-5585
Qubit dsDNA Quantification Assay Kits,  cat no. Q32851
MiSeq Reagent Kit v2 50 cycles (PE), Illumina cat no. MS-102-2001

mORF TF-wide screen
Day -1: CD8+ T cells are isolated and stimulated with anti-CD3-CD28 Dynabeads (see Isolation and culture of primary human T cells, Ref 1).
Day 0: 24 hours after isolation and stimulation, CD8+ T cells are transduced (n=3 biological replicates) with the mORF pooled lentiviral library (Ref 2, Addgene #192821) at 10% v/v and expanded for 3 days in standard media.
Day 3: Cells are selected with puromycin at 1 µg/mL for two days.
Day 5: Dynabeads are removed, and cells are split into two conditions: expanded and exhausted. Both conditions are supplemented with 0.5 ug/mL of puromycin, and the exhaustion condition is additionally restimulated with fresh dynabeads at a 3:1 concentration. 
Day 7: Both conditions are replenished with standard media with no puromycin. Dynabeads are removed and replenished for the exhaustion condition at a 3:1 concentration.
Day 9: Expanded cells are fixed and permeabilized following manufacturer protocol (FOXP3 TF staining kit, Thermo, cat no. 00-5523-00), and stained for TOX (Miltenyi Biotec, cat no. 130-118-335) at 1:100 dilution.
Day 10: Exhausted cells are fixed/permed following manufacturer protocol (FOXP3 TF staining kit, Thermo, cat no. 00-5523-00), and stained for TOX (Miltenyi Biotec, cat no. 130-118-335) at 1:100 dilution. 
Cells from each condition are sorted for top and bottom 10% TOX expressing cells using an SH800 FACS Cell Sorter (Sony Biotechnology). Genomic DNA is isolated from the sorted cell populations for mORF barcode library construction and sequencing. All replicates are maintained and sorted at a minimum of 100x coverage.
Note: Cells are maintained at 1–2 × 10 (Ref 3) cells ml−1 for all expansion steps unless otherwise indicated.
Genomic DNA isolation, mORF barcode PCR and sequencing mORF libraries
Genomic DNA is isolated using the PicoPure DNA extraction kit (Applied Biosystems, cat no. KIT0103) following manufacturer recommendations and incubated at 65 C overnight for reverse crosslinking. 
Genomic DNA is split across 100 μl PCR reactions (22 cycles at 98 °C for 10 s, 60 °C for 10 s, and 72 °C for 25 s) with NEBNEXT 2× Master Mix (NEB, cat no. M0541L) and up to 1 μg of genomic DNA per reaction.
PCRs from each samples are pooled together and purified using double-sided magnetic Ampure XP bead selection (Beckman Coulter, cat no. A63882) at 0.5× and 1.0×.
Libraries are run on a High Sensitivity D1000 tape (Agilent, cat no. 5067-5584) to confirm amplicon size and quantified using Qubit’s dsDNA High Sensitivity assay (Invitrogen, cat no. Q32851).
Libraries are diluted to 2 nM, pooled together at equal volumes, and sequenced using MiSeq Reagent Kit v2 50 cycles (Illumina, cat no. MS-102-2001).
Oligo Table (Ref 1)
ABCD
MethodOligo NameOligo Sequence
MORF ScreenTF_mORF_NGS_FWAATGATACGGCGACCACCGAGATCTACACTAACTTGAAAGTATTTCGATTTCTTggAGCTAttggctttatatatcttgtggaaaggacga
MORF ScreenTF_mORF_NGS_BC_REV1. CAAGCAGAAGACGGCATACGAGATTCGCCTTGGTGACTGGAGTTCAG
ACGTGTGCTCTTCCGATCTTAAAGCAGCGTATCCACATAGCGT
MORF ScreenTF_mORF_NGS_BC_REV2. CAAGCAGAAGACGGCATACGAGATATAGCGTCGTGACTGGAGTTCAGA
CGTGTGCTCTTCCGATCTTAAAGCAGCGTATCCACATAGCGT
MORF ScreenTF_mORF_NGS_BC_REV3. CAAGCAGAAGACGGCATACGAGATGAAGAAGTGTGACTGGAGTTCAG
ACGTGTGCTCTTCCGATCTTAAAGCAGCGTATCCACATAGCGT
MORF ScreenTF_mORF_NGS_BC_REV4. CAAGCAGAAGACGGCATACGAGATATTCTAGGGTGACTGGAGTTCAGA
CGTGTGCTCTTCCGATCTTAAAGCAGCGTATCCACATAGCGT
MORF ScreenTF_mORF_NGS_BC_REV5. CAAGCAGAAGACGGCATACGAGATCGTTACCAGTGACTGGAGTTCAGA
CGTGTGCTCTTCCGATCTTAAAGCAGCGTATCCACATAGCGT
MORF ScreenTF_mORF_NGS_BC_REV6. CAAGCAGAAGACGGCATACGAGATGTCTGATGGTGACTGGAGTTCAGA
CGTGTGCTCTTCCGATCTTAAAGCAGCGTATCCACATAGCGT
MORF ScreenTF_mORF_NGS_BC_REV7. CAAGCAGAAGACGGCATACGAGATTTACGCACGTGACTGGAGTTCAGA
CGTGTGCTCTTCCGATCTTAAAGCAGCGTATCCACATAGCGT
MORF ScreenTF_mORF_NGS_BC_REV8. CAAGCAGAAGACGGCATACGAGATTTGAATAGGTGACTGGAGTTCAGA
CGTGTGCTCTTCCGATCTTAAAGCAGCGTATCCACATAGCGT
MORF ScreenTF_mORF_NGS_BC_REV9. CAAGCAGAAGACGGCATACGAGATTCCTTGGTGTGACTGGAGTTCAGA
CGTGTGCTCTTCCGATCTTAAAGCAGCGTATCCACATAGCGT
MORF ScreenTF_mORF_NGS_BC_REV10. CAAGCAGAAGACGGCATACGAGATACAGGTATGTGACTGGAGTTCAGA
CGTGTGCTCTTCCGATCTTAAAGCAGCGTATCCACATAGCGT
MORF ScreenTF_mORF_NGS_BC_REV11. CAAGCAGAAGACGGCATACGAGATAGGTAAGGGTGACTGGAGTTCAG
ACGTGTGCTCTTCCGATCTTAAAGCAGCGTATCCACATAGCGT
MORF ScreenTF_mORF_NGS_BC_REV12. CAAGCAGAAGACGGCATACGAGATAACAATGGGTGACTGGAGTTCAG
ACGTGTGCTCTTCCGATCTTAAAGCAGCGTATCCACATAGCGT
Oligo Table

Protocol references
1. Savage RE, McRoberts Amador CD. et al. Transcription factor collaboration enables precise T cell state engineering. bioRxiv 2026.04.20.718569.  http://biorxiv.org/content/early/2026/04/22/2026.04.20.718569

2. Joung,  J. et al. A transcription factor atlas of directed differentiation. Cell 186, 209–229.e26 (2023). 10.1016/j.cell.2024.04.038

3. Kartha,  V. K. et al. Functional inference of gene regulation using single-cell multi-omics. Cell Genom. 2, 100166 (2022). https://doi.org/10.1016/j.xgen.2022.100166