Comment[GEAAccession]	E-GEAD-647
MAGE-TAB Version	1.1
Investigation Title	Genome-wide CRISPR screen in human T cells reveals regulators of FOXP3 [inChIP-seq]
Experiment Description	Regulatory T cells (Tregs), which specifically express the master transcription factor FoxP3, play a pivotal role in maintaining immunological tolerance and homeostasis and have the potential to revolutionize cell therapies for autoimmune diseases. While stimulation of naive CD4+ T cells in the presence of TGF-beta and IL-2 can induce FoxP3+ Tregs in vitro (iTregs), the resulting cells are often unstable and have thus far hampered translational efforts. A systematic approach towards understanding the regulatory networks that dictate Treg differentiation could lead to more effective iTreg cell-based therapies. Here we performed a genome-wide CRISPR loss-of-function screen to catalog gene regulatory determinants of FOXP3 induction in primary human T cells and characterized their effects at single-cell resolution using Perturb-icCITE-seq. Our findings reveal novel regulators of FOXP3 and point towards new avenues to improve the efficacy of adoptive cell therapy for autoimmune disease.
Experimental Design	genetic modification design
Experimental Factor Name	cell_type	antibody	genetic_modification
Experimental Factor Type	cell_type	antibody	genetic_modification
Person Last Name	Takeshima
Person First Name	Yusuke
Person Affiliation	Osaka University, WPI Immunology Frontier Research Center
Person Roles	submitter
Public Release Date	2025-01-07
PubMed ID	40140585
Protocol Name	P-GEAD-1993	P-GEAD-1994	P-GEAD-1995	P-GEAD-1996	P-GEAD-1997
Protocol Type	sample collection protocol	nucleic acid extraction protocol	nucleic acid library construction protocol	nucleic acid sequencing protocol	normalization data transformation protocol
Protocol Description	T cells were subjected to FOXP3 induction by stimulation for 72 hours using Dynabeads Human T-Activator CD3/CD28 (Thermo Fisher, #11131D) at a 1:1 ratio and supplementation  with 500 IU/mL of hIL-2 (R&D Systems), 5 ng/mL of hTGF-beta (R&D Systems), 10 ug/mL of Ascorbate (Sigma-Aldrich), 10 nM of ATRA (Sigma-Aldrich), 5 ug/mL of anti-hIFN-gamma, -hIL-4, -hIL-6 and -hTNF-alpha (BioLegend) antibodies and 5 ug/mL of anti-FasL (BioLegend) antibodies. Cultured T cells were sorted after stimulation using BD FACSAria Fusion or FACSAriaIII to enrich for purified cells.	For each reaction, 30 ul Dynabeads Protein G and Protein A magnetic beads (1:1 ratio; Invitrogen) were resuspended in 500 ul bead wash buffer (0.5% BSA in PBS) and then pre-incubated with approximately 3 ug of the appropriate antibody at 4C on a rotator for at least 2 hours. Subsequently, the reactions were placed on a magnet and washed three times with 500 ul bead wash buffer to remove excess antibodies and then resuspended to the original bead volume with bead wash buffer.  Fixed cells were sorted and resuspended in 100 ul nuclear lysis buffer (50 mM Tris-HCl pH 7.5, 10 mM EDTA, 1% SDS), supplemented with 1x protease inhibitor cocktail (Roche, 11873580001) and incubated on ice for 10 minutes. Sonication was performed on the Picorupter platform (Diagenode) for 5 cycles of 30s ON/30s OFF. Sheared chromatin was centrifuged at 10,000 g, 10 minutes, 4C and the clarified supernatant was transferred to a new DNA LoBind Eppendorf tube (Eppendorf). Chromatin was diluted 10x using ChIP dilution buffer (16.7 mM Tris pH 7.5, 167 mM NaCl, 1.2 mM EDTA, 1.1% TritonX-100, 0.01% SDS) supplemented with 1x protease inhibitor cocktail and blocked with 5 ul Fab Fragment Goat Anti-Mouse IgG (Jackson ImmunoResearch, 115-007-003) on ice for at least one hour, with occasional mixing by inversion. The reaction was incubated overnight at 4C on a rotator with antibody-conjugated beads. On the next day, beads were washed and reverse-crosslinked with the iDeal ChIP-seq kit for Histones (Diagenode; C01010173) as per manufacturer's recommendations and then column purified with ChIP DNA Clean & Concentrator (Zymo Research; D5205).	Library preparation was performed with the KAPA HyperPrep kit (Roche; KK8504) according to manufacturer's instructions.	Prepared libraries were sequenced on the NovaSeq platform (Illumina).	Fastq files were aligned to the human genome (GRCh38; GenBank assembly GCA_000001405.40) using bowtie2. Genomic tracks were generated using the deepTools bamCoverage function under default settings.
SDRF File	E-GEAD-647.sdrf.txt
Comment[AEExperimentType]	ChIP-seq
Comment[BioProject]	PRJDB16517
Comment[Last Update Date]	2025-08-13