Chip kit

ChIP assay was conducted using a ChIP kit (abcam, ab500) following the manufacturer’s guidelines. Briefly, CD4+ T cells isolated from TNBS-induced IBD mice administrated with fungi were fixed, lysed, and sonicated. Then, the cell lysates were incubated with NF-κB p65 (CST, #8242) or immunoglobulin G (IgG). Finally, the GLS2 DNA was detected using RT-PCR.
Quantitative real-time PCR
CD4+ T cells were collected and lysed in TRIzol Plus (Takara) to extract total RNA, and 500 ng total RNA was reverse transcribed into cDNA using PrimeScript RT Master Mix (Takara). The cDNA was further mixed with SYBR Green PCR Master Mix and specific primers (Takara) to detect relative mRNA expression of specific genes.

After the stimulation, chromatin immunoprecipitation (ChIP) assay was performed using a ChIP kit (Abcam) as described by the manufacturer’s instruction. In brief, chromatin from crosslinked macrophages was sheared by sonication (8 of 30 s-on and 30 s-off pulses at high power output, Bioruptor Plus, USA) and incubated overnight with rabbit anti-Phospho-Histone H3S10 (Abcam) at 4 °C. Precipitated DNAs were analyzed by RT-qPCR with SYBR Green Premix Ex Taq (Takara) and primers for human IL-8 promoter (−121 to +61) 5′-GGGCCATCAGTTGCAAATC-3′ and 5′-TTCCTTCCGGTGGTTTCTTC-3′, human IκBα promoters (−316 to −15) 5′-GACGACCCCAATTCAAATCG-3′ and 5′-TCAGGCTCGGGGAATTTCC-3′, as well as a human β-actin promoter (−980 to −915) 5′-TGCACTGTGCGGCGAAGC-3′ and 5′-TCGAGCCATAAAAGGCAA-3′.

Chromatin immunoprecipitation (ChIP) assay was performed using the ChIP kit (Abcam, AB500). The cells were lysed and sonicated to obtain appropriate lengths of DNA fragments, and a small part of the chromatin that had been sonicated was taken to determine the length of DNA fragments. The DNA samples were quantitatively divided into three samples, including the tested group, positive control, and negative control, and the remaining DNA samples were used as the Input groups. Three samples were incubated with the corresponding antibodies and microbeads for immunoprecipitation overnight. The DNA purification was performed to obtain the DNA fragments bound to the target antibodies. Purified DNA was used as the template to amplify target fragments (Primers for p65: forward, 5′-GTACCGAGAAGACATAAATCGCT-3′; Reverse, 5′-TAACAAGGAAGACTGGATTTGTCT-3′. Primers for c-Fos: forward, 5′-GCTATGAGTGTTACAGAGGGGG-3′; Reverse, 5′-TACTTCCCCTCATTCTGGCCG-3′), and the amplified DNA fragments were analyzed by the agarose gel electrophoresis.

All the primers used in this study were designed using Primer3 (http://bioinfo.ut.ee/primer3–0.4.0/) and based on D. Melanogaster assembly Apr. 2006 (BDGP R5/DM3).
Millipore's ChIP Kit was used for performing ChIP with H3K4me3 antibody (Abcam) as described above. S2 cells transfected with wt and T91A were used. The list of primers used for quantitative PCR is given in Supplementary Table 3. To obtain the fold depletion, enrichment value obtained from cells transfected with wt BEAF 32B was divided by the enrichment value obtained from cells transfected with T91A mutant BEAF 32B.

Chromatin immunoprecipitation (ChIP) assay was performed using the ChIP kit (Abcam, Waltham, MA, USA; ab500) according to the supplied protocol. Briefly, HEK293FT cells were exposed to 5 μg·mL⁻¹ tunicamycin (Sigma‐Aldrich) for 5 h, cross‐linked with 1.1% formaldehyde (Thermo Scientific, Waltham, MA, USA; Cat# 28906) for 10 min at room temperature, and quenched in 0.125 m glycine. The cells were then incubated with lysis buffer and sonicated to produce 200–500 base pair DNA fragments. DNA fragments were immunoprecipitated from the cell lysates using anti‐ATF4 antibody (Abcam; ab184909) or rabbit IgG (Abcam; ab171870), and immunoprecipitates were recovered by the addition of DNA purifying slurry. After reverse crosslinking and washing, purified DNA was quantified by SYBR Green real‐time PCR (Bio‐Rad, Hercules, CA, USA) using specific primers (Table 2). The samples added rabbit IgG was used as a control. Data were expressed as the percentage of input.

We used approximately one million HeLa cells per reaction. Chromatin shearing was performed using a sonicator (Branson). We carried out the ChIP using the ChIP Kit (Abcam, Cambridge, UK #ab500) following instructions of the manufacturer. For the pull-down step, the following antibodies and quantities were used: anti-Histone H3 antibody as positive control (Abcam, #ab1791; 2.5 μg), a Rabbit-anti-Mouse-Alexa Fluor 488 (ThermoFisher, Waltham, USA, #A-11,059; 5 μg), and an anti-PPARγ (Abcam, #ab59256; 5 μg). After DNA purification, we used 2 μl of DNA for qPCR, we carried out the reaction with the EDN1prom primers from Supplementary Table S3 using Power-Up SYBR Green Master Mix (ThermoFisher, Waltham, USA). For data analysis, we used the input percent method [100 × 2^{(InputCT−CT(IP)}].