Bioruptor ucd 300

Purified cells were fixed with 1% formaldehyde (Sigma) at a concentration of approximately 10⁶ cells/ml. Fixed cell preparations were sonicated using a Diagenode Bioruptor UCD-300 for 3x10 min (30 s on; 30 s off). 67 μl of chromatin (1 million cells) were incubated with 229 μl dilution buffer, 3 μl protease inhibitor cocktail and 0.5–1 μg of H3K4me3 antibody (Diagenode) and incubated overnight at 4°C with rotation. Protein A/G magnetic beads were washed in dilution buffer with 0.15% SDS and 0.1% BSA, added to the chromatin/antibody mix and rotated for 60 min at 4°C. Beads were washed with 400 μl buffer for 5 min at 4C with five rounds of washes. After washing chromatin was eluted using elution buffer for 20 min. Supernatant was collected, 8 ml 5M NaCl, 3 ml proteinase K were added and samples were incubated for 4 h at 65°C. Finally, samples were purified using QIAGEN; Qiaquick MinElute PCR purification Kit and eluted in 20 ml EB.

Cells (5 × 10⁶ cells per assay) were cross-linked with 1% formaldehyde (Sigma; F8775), quenched with 0.125 M glycine, and then sonicated using a Bioruptor UCD-300 (Diagenode) to yield chromatin fragments. Then 100 μg of lysate was immunoprecipitated with anti-IgG (negative control), anti-PRMT5 (Sigma; P0493), anti-H4R3me2s (Abcam: ab5823), or anti-H3R8me2s (Abcam; ab272149) antibodies immobilized on protein A-Sepharose beads (Sigma; P3391). The immunoprecipitated complexes were washed sequentially with washing buffer (10 mM Tris–HCl, 500 mM NaCl, 1% Trition X-100, 0.1% SDS, 0.5% Na-deoxycholate). After digestion with proteinase K (Beyotime; ST532), the DNA fragments were extracted with phenol:chloroform:isoamyl alcohol (25:24:1 v:v:v) and precipitated with 100% ethanol at -20 °C in the presence of 0.3 M sodium acetate (pH 5.2) and 2 µg of glycogen (Beyotime; D0812). Finally, the glycogen-protein pellet was suspended in H₂O and the purified DNA was subjected to real-time PCR analyses. The ChIP primer sequences are listed in Supplementary Table S4.

Primary microglial cells were treated with OGD and FTY720, and Chromatin immunoprecipitation (ChIP) assay was performed to evaluate the acetylation levels of histone H3 and H4 binding to the Sp1 in the rat Klf4 promoter region as described previously. Briefly, cells were cross-linked in 37% formaldehyde for 10 min and the reaction was stopped by adding a final concentration of 0.125 mM of glycine and incubated for 5 min. After homogenizing the sample in cell lysis buffer the nucleus phase was pelleted and then suspended in nucleus lysis buffer. Samples were then sheared by sonication (Bioruptor UCD-300, Diagenode) and the chromatin concentration and chromatin fragments were checked using Nanodrop and 2% agarose gel. Then, samples were immunoprecipitated using histone H3- and H4-antibody linked to protein A beads (Santa Cruz) and the DNA purified with Phenol/chloroform extraction. The relative abundance of the histone H3- and H4-antibody precipitated chromatin containing the Sp1 binding site in the rat Klf4 promoter region was detected by qPCR. Site of KLF4 promoter located between −859 and −868 from the transcription starting site (Figure 5B), and the following primer set for promoter quantification: Sense: CGTGTGACCTTGCGATAG, Antisense: CTCTATGAGTGCGTAGGATG.

ChIP-seq was done with minor modifications from a published protocol [13 (link)]. Briefly, the cells grown on 10 cm dishes were fixed with formaldehyde at room temperature (final concentration 1 % v/v; 8 min for K562 cells and 10 min for VCaP cells), and cross-linking was stopped with glycine (final concentration 125 mM). Chromatin was fragmented to approximately 200 to 500 bp using sonication (Bioruptor UCD-300, Diagenode). Target antibodies (Ab) were coupled to protein-A or -G beads (Millipore), fragmented chromatin was incubated with Ab-coupled beads overnight, washed, eluted, and de-cross-linked in the presence of proteinase K (Fermentas). Chromatin fragments were purified using MinElute columns (Qiagen), ChIP-seq libraries were prepared using NEBNext kit (New England Biolabs) and sequenced at BGI (Hong Kong, China; K562 SUMO2/3 control and HS; VCaP Pol2 control) or at EMBL genomics core facility (Heidelberg, Germany; all other samples). Two biological replicates were made for each sample. Fragmented de-cross-linked chromatin was used as a control for K562 samples and previously published IgG ChIP-seq chromatin for VCaP samples [15 (link)].

Chromatin immunoprecipitation followed by next generation sequencing (ChIP-seq) was performed as previously described [11 (link)]. Briefly, 100 mg of VAT was cross-linked after what nuclei were purified. Then, nuclei were lysed, and the chromatin was sheared by sonication using a Bioruptor UCD-300 (Diagenode, Liège, Belgium). A total of 5 μg of chromatin were used for immunoprecipitation, which were performed by incubating the chromatin with 1.5 μg of anti-H3K4m3 (ab8580, abcam) fixed to protein G-coated magnetic beads (Dynabeads Protein G; Thermo Fisher Scientific, Carlsbad, CA, USA) at 4 °C overnight. Chromatin was released and purified (MinElute PCR Purification Kit, Qiagen, Hilden, Germany) and DNA samples were sequenced using Hiseq4000 (Illumina, San Diego, CA, USA) and paired-end mode (2 × 100 bp).

ChIP experiments with C2C12 cells were carried out with the MAGnify™ Chromatin Immunoprecipitation System (Life Technologies) following the manufacturer's instructions with some modifications. Sonication was performed using the Bioruptor UCD300 (Diagenode) to obtain chromatin fragments of approximately 100–500 bp. For Hey1 ChIP, C2C12 cells were transiently transfected with Flag-Hey1 and empty Flag vector, which was used as a control. Anti-pDPF3a and anti-Flag M2 antibodies were used in ChIP experiments. ChIP DNA was purified using the Zymo ChIP DNA cleanup kit. ChIP results were analyzed by quantitative PCR. ChIP enrichment was normalized to the input and expressed as relative enrichment of the material precipitated by the indicated antibody on target regions. ChIP primers are listed in Supplementary Table S4.