Dna purification kit

An EZ-ChIP™ kit (Millipore) was applied for the ChIP assay. Methanol was used to fix cells, which was stopped by glycine. Cells were scraped and centrifuged to obtain cell precipitates. The precipitates were suspended with cell lysis encompassing phenylmethylsulphonyl fluoride and centrifuged, followed by the removal of supernatants. DNA was broken in an ice-water bath. Afterward, 10% of the supernatants were used as control and the rest 90% of the supernatants were incubated with magnetic beads coupled with H3K4me3 (ab213224, Abcam) or normal rabbit IgG (NC) antibodies, followed by centrifugation. DNA bindings to H3K4me3 were eluted with eluent and purified with a DNA purification kit (Beyotime, Shanghai, China). Lastly, immunoprecipitated DNA was quantified with qRT-PCR.

ChIP assays were performed following the instructions provided with the ChIP assay kit (Beyotime, Wuhan, China). First, the chromatin in AR42J cells was cross-linked with 1% formaldehyde for 10 min at 37 °C, and then the cells were washed three times with cold PBS. The cells were collected, lysed, and sonicated. The nuclear lysates were sonicated, and an equal amount of chromatin was immunoprecipitated at 4 °C overnight with 3 μg of GR and IgG anti-rat monoclonal antibody (CST, United States). The immunoprecipitated products were collected after incubation on protein A + G-coated magnetic beads; then, the beads were washed, and the bound chromatin was eluted in ChIP elution buffer. The protein was digested with proteinase K for 4 h at 45 °C. The DNA was purified using a DNA purification kit (Beyotime). The DNA fragments of the GR binding sites in the miR-22 promoter were designed and synthesized by RiboBio (Guangzhou, China). After immunoprecipitation, the GR binding site was evaluated using qPCR and normalized to the total chromatin. Total chromatin was used as the input. IgG and a non-specific GR binding site (Nbs) were used as controls. The primers used to amplify the DNA fragments of the GR binding sites in the miR-22 promoter were also designed and synthesized by RiboBio (Guangzhou, China). The ChIP-qPCR conditions were based on a three-step method.

The primers used to clone Tp2FT included the following: 5′- CATATGATATTATTTTGGTC-3′ and 5′-GGATCCTTATTT ATCAATAACGAAGG-3′. A 50 μL PCR reaction included the following: 10 × Taq polymerase Buffer (5 μL), template DNA (1 μL), forward and reverse primers (1 μL each), dNTPs (1 μL), ddH₂O (40.5 μL), Taq polymerase (0.5 μL). The PCR conditions were as following: initial denaturation for 5 min at 94℃, followed by 30 cycles of denaturation for 1 min at 94℃, annealing for 40 s at 52℃, and extension for 2 min and 30 sec at 72℃, and final extension at 72℃ for 5 min. The PCR product and the vector plasmid pET15b were digested with NdeI and BamHI restriction enzymes and purified using the DNA Purification Kit (Beyotime, Shanghai, China). The recombinant expression vector was obtained by the ligation of the target gene with vector in the presence of DNA ligase. The recombinant plasmid was identified using DNA sequencing and a macro-restriction map. The screened positive plasmids were transformed into E. coli BL21 (DE3) chemically competent cells. After obtaining the recombinant expression vector, it was transferred into E. coli, which was grown on ampicillin medium.