Bioruptor pico sonication device

7–10 × 10⁶ NF or CAF at subconfluency were fixed in 1% formaldehyde and sonicated using the Bioruptor Pico sonication device (Diagenode; B01060001) using 15 cycles (30 s on; 30 s off) at maximum intensity. Purified chromatin was then separated for: (i) immunoprecipitation using Dynabeads Protein G (Life Technologies: 10003D) coated with 8 µg and 4 µg of HSF1 antibody (ThermoFisher: RT-405-P, and Cell Signalling: 4356 S, respectively) per ChIP experiment; (ii) non-immunoprecipitated chromatin, used as Input control; and (iii) assessment of sonication efficiencies using a 1% agarose gel. For the quantitative PCR briefly, reactions were carried out in 10 μL volume containing 5 μL of Sybergreen mix (Applied Biosystems; 4472918), 0.5 μL of primer (5 μM final concentration), 2.5 μL of genomic DNA and 2 μL of DNAse/RNAse-free water. A three-step cycle programme and a melting analysis were applied. The cycling steps were as follows: 10 s at 95 °C, 30 s at 60 °C and 30 s at 72 °C, for 40 cycles. Enrichment of the immunoprecipitated sample was confirmed using positive and negative controls. The exact loci of the primers are as follows: negative control (gene desert): chr6:116908976–116909177; Dkk3 Promoter (P): chr7:112159485–112159638; Dkk3 Enhancer (E): chr7:112183116–112183344; Rilpl, within the gene (positive control): chr5:124510011–124510190.

Recombinant full-length tau (tau^2N4R) was as described in previous protocols⁵⁷ . Lyophilised protein aliquots were freshly dissolved in 10 mM HEPES, pH 7.4, 100 mM NaCl at a final concentration of 10 μM. Following filtration, using 0.2 μM PVDF filters, 5 μM of heparin (Sigma) was added to the solution to induce aggregation. After 7 days of incubation at 37 °C under shaking (700 rpm), we generated seeds by breaking endpoint amyloid fibrils through successive sonication for 15 min (30 s on, 30 s off) at 10 °C, using a Bioruptor Pico sonication device (Diagenode).

The individual Deschampsia cespitosa used for this analysis was collected in a clearing of a spruce forest in the Karawanken mountain range, Carinthia, Austria (a voucher specimen is deposited in the herbarium WU). Leaves were dried in silica gel and DNA was extracted from 20 mg of dry tissue with the DNeasy Plant Mini Kit (Qiagen, Hilden, Germany), following the manufacturer’s protocol. The total DNA was visualized with agarose gel electrophoresis on a transilluminator Gel Doc 2000 (Biorad, Vienna, Austria) while its quality and quantity were assessed using a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, Vienna, Austria) and a Qubit 2.0 (Thermo Fisher Scientific). Whole cellular DNA (nuclear, mitochondrial and plastid DNA) was sheared with a Bioruptor^® Pico sonication device (Diagenode, Liege, Belgium) using seven cycles of 15 s on and 90 s off at 4 °C in order to obtain fragments with an average size of 350 to 400 bp. Fragment size was checked afterwards with an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). The library was prepared with a TruSeq DNA PCR Free Library Kit (Illumina, San Diego, CA, USA), following the manufacturer’s protocol and barcodes provided with the kit. The library was sequenced in a 1/24 lane of an Illumina HiSeq 3000 at VBCF Vienna (https://www.vbcf.ac.at/facilities/next-generation-sequencing/).

Cell lysates were prepared in ice-cold RIPA lysis buffer (50 mM Tris pH 8, 150 mM NaCl, 1% NP40, 0.5% Na-deoxycholate, 0.1% SDS) supplemented with HALTTM Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific) and sonicated using a Bioruptor^® Pico sonication device (diagenode). Protein content was determined with the BCA Protein Assay Kit (Pierce). 20 µg of protein were resolved by SDS–PAGE, transferred to Nitrocellulose membrane (Thermo Fisher Scientific) and blocked in 5% Milk/TBST for 1 h at RT. Primary antibodies were used as follows: anti-p63 (ab735; 1:500; abcam), anti-phospho FAK-Y397 (#8556; 1:500; Cell Signaling Technology), anti-FAK (#3285; 1:1000; Cell Signaling Technology), anti-phospho Src-Y416 (#6943; 1:1000; Cell Signaling Technology), anti-Src (#2109; 1:1000; Cell Signaling Technology), TCF7 (#2203; 1:1000; Cell Signaling Technology), anti-GAPDH (#5174; 1:5000; Cell Signaling Technology), and anti-ACTB (#A2228; 1:1000, Sigma-Aldrich).

HCT116 cells and RKO cells were fixed in 1% paraformaldehyde for 8 min, and ChIP was performed using the iDeal ChIP-seq kit (Diagenode, Liege, Belgium) according to the manufacturer’s instructions. Chromatin extracts from one million cells were sonicated to yield 100 to 500 bp fragments using a Bioruptor Pico sonication device (Diagenode) on high power for two rounds of 6 cycles with 30 s on 30 s off at 4 °C. Fragmentation was verified with agarose gel electrophoresis. ChIP-seq was performed using 1 μg of H3K4me1 antibody (ab8895, Abcam) per ChIP. After reverse cross-linking overnight at 65 °C, DNA was extracted using the Qiagen MinElute PCR purification kit in 15 μl of elution buffer. Libraries were prepared for sequencing using the NEBNext® ChIP-Seq Library Prep Reagent Set for Illumina® and sequenced on a HiSeq 2500 System (Illumina).

For each cell line, 8 × 10⁶ cells were counted as previously described, and centrifuged at 1500 rpm for 10 minutes at 4°C. The supernatant was removed, and the pellet was resuspended in ice cold PBS, before being centrifuged again for a further 10 minutes. Ice cold Pierce RIPA buffer (Thermo Scientific) prepared with Halt protease inhibitor cocktail (Thermo Scientific) and 0.5 M EDTA (Thermo Scientific) was added to each cell pellet and incubated on ice for 10 minutes. Each sample was sonicated for 3 cycles (30 seconds on, 30 seconds off) using a Bioruptor Pico sonication device (Diagenode) at 4°C, vortexing after the second cycle. Finally, each sample was centrifuged once more at 14000 rpm for 10 minutes at 4°C to remove any remaining insoluble material and the supernatant was aliquoted. Cell lysates were stored at -80°C until required.