Biorad s3

All steps were performed at 4 °C to ensure integrity of chromatin and RNA. For nuclear RNA isolation RNAsin (80 U/ml, Promega) was added to all buffers. Isolated nuclei were stained in 500 µl staining buffer (phosphate-buffered saline (PBS) containing 1 % bovine serum albumin (BSA), 22.5 mg/ml glycine, 0.1% Tween 20) using anti-PCM1 (1:500, HPA023370, Sigma) and anti-PLN antibodies (1:500, A010–14, Badrilla) for 30 min. For isotype control stainings, we used primary antibodies lacking target specificity (1:1000, anti-mouse, 554121, BD; 1:1000, anti-rabbit, Z25308, Life technologies). Subsequently, the corresponding Alexa488- and Alexa568-labeled secondary antibodies (1:1000, A11029 and A11011, Invitrogen) were added. After 30 min of incubation, nuclei were pelleted by centrifugation (1000 × g, 5 min) and resuspended in 1 ml PBS containing 1 mM ethylenediaminetetraacetic acid (EDTA). Nuclei were filtered (CellTrics 30 µm, Sysmex) and incubated with Draq7 (final concentration 2.25 nM, Cell Signaling) for 10 min. Nuclei were analyzed (Bio-Rad S3, Bio-Rad; LSRFortessa, BD) and sorted by flow cytometry (Bio-Rad S3, Bio-Rad).

Treg cells were isolated from total skin LNs of naïve mice or from inguinal and axillary dLNs of EAE mice (day 10 after immunization). LNs were scratched and LN cells were subjected to CD4⁺ T cell enrichment using a CD4⁺ T cell isolation kit (Miltenyi biotec). CD4⁺ CD25^hi Treg cells were next sorted using a MoFlowAstrios (Beckman Coulter) or a Biorad S3 (Biorad).

For CRISPR screening experiments, K562 cells were passaged to maintain cell density between 500,000 and 2 million cells/ml. Cells were propagated in IMDM + 10% FBS + penicillin/streptomycin + appropriate antibiotics (blasticidin 5 μg/ml, zeocin 50 μg/ml) until 200 million cells were obtained (approximately 8–10 d). For infection, 200 million cells were pelleted and resuspended in IMDM + 10% FBS + 8 μg/ml polybrene. Date of infection was day 0. An MOI of 0.4 was used to minimize multiple infection events per cell. Cells were infected overnight, pelleted, and exchanged into fresh media. After 24 h, cells were split, and 2 μg/ml puromycin was added. Cells were continually passaged in puromycin. At day 10, cells were removed from puromycin, and at day 12, cells were sorted for Red:Green fluorescence. The amount of 100 M unsorted cells were pelleted and processed as input. The top and bottom 30% of cells (based on Red:Green ratio) were taken; 100 million cells were sorted for each experimental condition. Cell sorting was performed using a FACSAria (Becton Dickinson, Franklin Lakes, New Jersey) or BioRad S3 (Bio-Rad, Hercules, CA) sorter. Cells were pelleted and stored at −80°C until processing.