Tbe page gels

Indel formation was tested in HEK293T cells by the T7EI assay. HEK293T cells were cultured in DMEM high glucose medium (GIBCO) supplemented with 10% FBS, 1X L-glutamine, 1X MEM-NEAA, and 1X penicillin/streptomycin (Life Technologies) in 37°C incubators under 5% CO2. Cells cultured in 12-well plates to 60%–80% confluence were transfected with 0.5 μg CAGGS-AsCpf1-U6-INS-sgRNA plasmids using FuGENE 6 transfection reagent (Promega) based on the manufacturer’s guideline. Two to three days post transfection, cells were dissociated with trypsin, and GFP positive cells were sorted on a FACSAria (BD Biosciences), followed by DNA purification with DNeasy Blood & Tissue Kit (QIAGEN). For T7EI assay, PCR reactions were performed with 0.5 μg genomic DNA as template, and PCR with AmpliTaq Gold DNA Polymerase (Life technology) with the following primers:
INS: 5′-TGGGGCAGGTGGAGCTGGGCGG-3′, and 5′-ACCACCCCTGGCCCCTCAGAGACC-3′.
A total of 200 ng PCR products were annealed in NEB buffer 2 and processed with 5 units of T7EI (NEB) for 1 hour at 37°C before resolved on 10% TBE PAGE gels (BioRad) run at 100 V for about 2 hours. The PAGE gels were stained with ethidium bromide, and images were taken with an AlphaImager gel documentation system (Alpha Innotech). Mutation efficiency were measured as previously reported.⁶⁴ (link)

GR mutant-expressing cells were left untreated or treated with 100 nM of Dex (Sigma-Aldrich) for 1 h. For ChIP, after cross-linking with paraformaldehyde and cell collection, the chromatin was sonicated (Bioruptor, Diagenode) to an average DNA length of 200–500 bp. For immunoprecipitation, 600 µg of chromatin was incubated with antibody (for details, see Supplemental Methods) coupled onto Dynabeads magnetic beads (Thermo Fisher Scientific) with rotation overnight at 4°C. After stringent washes, the antibody-bound chromatin fragments were eluted, the cross-linking was reversed, and the remaining proteins were digested. DNA was extracted from the samples with phenol-chloroform extraction and ethanol precipitation. ChIP-seq libraries were generated using a TruSeq ChIP sample prep kit (Illumina IP-202-1012) according to the manufacturer's instructions. For ATAC, the cells were detached from the flasks using 5 mL of Accutase (Thermo Fisher Scientific) by incubating 5 min at RT. After nuclei isolation, ATAC was performed according to the published protocol (Buenrostro et al. 2015 (link)). Size selection was performed using SPRIselect (Beckman Coulter) to remove <150-bp and >800-bp fragments according to the manufacturer's instructions. Size selection was verified using 5% TBE PAGE gels (Bio-Rad).