Mar 30, 2026
TF Perturb-seq protocol
- Nan Zhang1,
- Michael Beer2,
- Danwei Huangfu1
- 1Sloan Kettering Institute;
- 2Johns Hopkins University
Protocol Citation: Nan Zhang, Michael Beer, Danwei Huangfu 2026. TF Perturb-seq protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l6emw5gqe/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: February 09, 2026
Last Modified: March 30, 2026
Protocol Integer ID: 242948
Keywords: ESC, DE, TF Perturb-Seq, Perturb-Seq, epigenetic regulators in embryonic stem cell, epigenetic regulator, embryonic stem cell, seq protocol tf perturb, definitive endoderm, seq protocol, targeting tf, seq, tf perturb, esc
Funders Acknowledgements:
NHGRI
Grant ID: U01 HG012051
Abstract
TF Perturb-seq protocol targeting TFs and epigenetic regulators in embryonic stem cells (ESC) and definitive endoderm (DE).
Troubleshooting
TF Perturb-seq protocol
We generated two sgRNA libraries: (i) a human transcription factor (hTF) library comprising 13,266 sgRNAs targeting 1,983 human TFs and epigenetic regulators, and (ii) a control library comprising 1,200 negative-control sgRNAs (600 non-targeting and 600 targeting). For the hTF library, six sgRNAs per transcript were selected from Replogle et al. (eLife, 2022), and an additional six lab-validated sgRNAs targeting GATA6 were included.
Each sgRNA library was transfected into 293T cells using JetPRIME (Cat. 89129-924, VWR) following the manufacturer’s instructions.
For lentiviral concentration, 100 mL of 1× lentiviral library supernatant was transferred into Ultra-Clear centrifuge tubes (Beckman Coulter; NC9146666) and centrifuged at 25,000 rcf at 4°C for 90 min. The supernatant was gently removed after centrifugation. Viral pellets were resuspended in 2 mL DPBS to generate a 50X-concentrated lentiviral library.
We targeted ~100× coverage per sgRNA for each Perturb-seq screen.
On Day 0, 4 million idCas9-KRAB SOX17-eGFP/+ HUES8 hESCs were infected with the 50X-concentrated hTF lentiviral library at an average MOI of 10 in 10-cm plates (1 million cells per plate).
In parallel, 1 million idCas9-KRAB SOX17-eGFP/+ HUES8 hESCs were infected with the 50X-concentrated control lentiviral library at an average MOI of 10 in one 10-cm plate.
Protamine sulfate (6 µg/mL per plate) was added during the first 24 h post-infection to enhance infection efficiency.
Infected cells underwent puromycin selection (3 µg/mL) for 3 days starting on Day 1 post-infection.
To induce dCas9-KRAB expression, doxycycline (2 µg/mL) was added to the culture media on Day 1 and maintained until the end of the screen (Day 11 for the ES condition and Day 14 for the DE condition).
On Day 7 post-infection, cells infected with the hTF and control libraries were pooled at a 19:1 ratio.
For the ES screen, pooled cells were maintained in E8 medium until Day 11. For the DE screen, pooled cells were expanded in E8 medium and then differentiated for 4 days to definitive endoderm (DE), with collection on Day 14.
For each screen, live single cells were isolated by flow cytometry after staining with LIVE/DEAD‱ Violet Viability/Vitality Kit (Invitrogen; L34958).
Sorted cells were processed for single-cell 3′ RNA-seq and sgRNA capture using the 10x Chromium Next GEM Single Cell 3′ HT Reagent Kit v3.1 (Dual Index).
Libraries were sequenced on an Illumina NovaSeq 6000.