Fagck001 1

Cells were cross-linked with 1% formaldehyde for 15 min and were lysed with 500 µl lysis buffer (20mM Tris HCl pH 8, 1% SDS, 50mM NaCl) containing 1x protease inhibitor cocktail (with EDTA). Chromatin samples were sheared to a length of 300-500 bp via sonication. Following centrifugation, supernatants were collected and 2 µg of antibody (#ab16645 [FOXP1], #ab17726 [FOXP4], Abcam) was added to chromatin to immunoprecipitate overnight. Dynabeads Protein G (10003D, Thermofischer) was added to antibody chromatin complexes for 2h. Next, protein G bead-chromatin complexes were washed twice with dilution buffer (20mM Tris HCl pH 7.6, 1% Triton X-100, 150mM NaCl) and once with washing buffer (PBS, 0.02% Tween-20, pH 7.4), as specified by manufacturer’s instructions. Proteinase K (P8107S, New England Biolabs) was added to immunoprecipiated and input chromatin; samples were then incubated at 45°C for 2h. Subsequently, chromatin was heated at 64°C for 4h for reversal of formaldehyde crosslinking. DNA fragments were purified using a PCR purification kit (FAGCK001-1, Favorgen) and were analyzed by qPCR. Antibodies used for ChIP are the same as for western blotting. Primer pairs used for ChIP assays are listed in Table S4.

To improve the yield and accuracy of sgRNA transcription, we modified a previously described method [40 (link)]. Briefly, sgRNA templates were generated by annealing two complementary oligonucleotides followed by PCR amplification. BamHI, BsaI, and KpnI restriction sites were attached to the ends of sgRNA templates with a second PCR. Tailed sgRNA templates were inserted into the pUC19 plasmid digested with BamHI and KpnI. sgRNA-encoding plasmids were linearized with BsaI, which resulted in proper sgRNA end sequences. Linearized plasmids were incubated with 7.5U/μl T7 RNA polymerase (NEB, M0251L) in reaction buffer (NEB, B9012S) containing 14 mM MgCl₂ (NEB, B0510A), 10mM DTT (Sigma, 43816), 0.02U/μl yeast inorganic pyrophosphatase (NEB, M2403L), 1U/μl murine RNase inhibitor (NEB, M0314L), 4mM ATP (NEB, N0451AA), 4mM GTP (NEB, N0452AA), 4mM UTP (NEB, N0453AA), and 4mM CTP (NEB, N0454AA) for 8 h at 37 °C. Yeast inorganic phosphatase was included to enhance sgRNA synthesis. After the reaction, the mixture was mixed and incubated with DNase I to remove the DNA template; transcribed sgRNAs were then purified using a PCR purification kit (Favorgen, #FAGCK001-1).