Simplechip enzymatic chromatin ip kit with magnetic beads

Chromatin immunoprecipitation (ChIP) assays were performed as reported previously (23 (link)) using untreated versus IFNα-treated HEL cells, untreated versus IFNα-treated control siRNA and ULK1 siRNA-transfected HEL cells, and vehicle (DMSO), SBI-0206965 (10 μmol/L) and/or IFNα-treated HEL cells, as indicated in the respective figure legends, and using the SimpleChIP Enzymatic Chromatin IP Kit with Magnetic Beads (#9003, Cell Signaling Technology) per manufacturer's instructions. HEL cells were transfected with either control siRNA or siRNAs targeting ULK1 using Amaxa Biosystems Nucleofector Kit V and program X-005 (Lonza) per the manufacturer's instructions one day prior to IFNα treatment. Anti-CHAF1B antibody (Proteintech, #27633-1-AP, RRID:AB_2880933) was used for IP of CHAF1B, and normal rabbit IgG (Cell Signaling Technology, #2729, RRID:AB_1031062) was used as negative control. qRT-PCR was performed on purified immunoprecipitated DNA for the ISG15 and IFIT1 promoter binding sites using SsoAdvanced Universal SYBR Green supermix (Bio-Rad) following manufacturer's guidelines with the following primers: ISG15 FWD 5′-CCA CTT TTG CTT TTC CCT GTC-3′, ISG15 REV 5′-AGT TTC GGT TTC CCT TTC CC-3′ and IFIT1 FWD 5′-GGT TGC AGG TCT GCA GTT TAT CTG T-3′ and IFIT1 REV 5′-AGC TGT GGG TGT GTC CTT GC-3′. All qRT-PCR signals were normalized to the input DNA.

ChIP assays were performed using SimpleChIP enzymatic chromatin IP kit with magnetic beads (9003, Cell Signaling Technology). The crosslinked chromatin was digested with micrococcal nuclease followed by sonication to break into 150–900 bp fragments. Immunoprecipitation was performed using anti-STAT3 (4904, Cell Signaling Technology) or Rabbit IgG. The enriched fragments were purified and analyzed by qPCR. The signal relative to input was evaluated using the formula as follows; percent input = 2% × 2^{(CT 2% input sample – CT IP sample)}, where CT indicates threshold cycle of qPCR reaction; IP, immunoprecipitation. The qPCR primers used are listed in Supplementary Table 5.

ChIP assay was performed with a p65 antibody 1:100 (Cell Signaling, #8242). The SimpleChIP Enzymatic Chromatin IP Kit with magnetic beads (Cell Signaling) was used according to the manufacturer's protocol. Briefly, TNFα treated (25 ng ml⁻¹) and untreated organoids were fixed with 1% formaldehyde for 10 min at room temperature. Glycine was added to quench cross-linking. After washing with ice-cold PBS, cells were lysed and digested with 5 μl micrococcal nuclease (2,000 gel units per μl) per sample for 20 min at 37 °C. Additionally, cells were sonicated to break the nuclear membrane. Lysates were incubated with a p65 antibody and Normal Rabbit IgG overnight at 4 °C. Protein G magnetic beads were added and samples were incubated for 2 h at 4 °C. Protein G magnetic bead pellets were washed with low- and high-salt washes. Next the chromatin was eluted from the antibody/Protein G beads and DNA was eluted with DNA elution buffer. The forward primer 5′-ACACTTTATGTGTGGTTAGAAAGGG-3′ and reverse primer 5′-ACCTGGATCTTTTCTAACAGGATG-3′ were designed to identify the Bcl-2 promoter region and used for PCR and quantitative real-time PCR analysis. PCR analysis was performed with Platinum Taq DNA Polymerase (Invitrogen) and Quantitative real-time PCR was performed with Sybr green (SensiFAST) according to manufacturer's protocol.