Bioruptor pico device

Immunoprecipitation reactions were carried out as described in (63 (link)). Proteins from dissected third instar larval CNS were extracted in cold PBS supplemented with cOmplete EDTA-free protease inhibitor cocktail (Merck). Tissues were crushed with a motorized pestle in a buffer containing 25 mM Hepes, 0.1 mM EDTA, 12.5 mM MgCl₂, 10% glycerol, 0.01% NP-40, 10 mM NaCl, and a cOmplete EDTA-free protease inhibitor cocktail before sonication with the Bioruptor pico device (Diagenode) (settings: 3 cycles, 30 s on/30 s off). Protein concentration was assayed by the DC Protein Assay (Bio-Rad), and 1 mg of proteins was used per reaction. Proteins were precleared with a 50:50 mix of protein A agarose beads (Sigma-Aldrich) and protein G Sepharose Fast Flow beads (Sigma-Aldrich) for 4 hours at 4°C. After bead elimination, samples were incubated with anti-Ph or anti-E(z) (1:200) overnight at 4°C. Beads were then added to the samples for 4 hours at 4°C. After three washes in the same buffer, proteins were boiled directly in 4× Laemmli sample buffer before being subjected to Western blot analysis (see below).

Chromatin immunoprecipitation (ChIP) was essentially performed as previously described (Antosz et al., 2020 (link); Michl-Holzinger et al., 2022 (link)). Plants (14-DAS in vitro grown) were crosslinked with formaldehyde and used for isolation of nuclei, before chromatin was sheared using a Bioruptor pico device (Diagenode). Immunoprecipitation was performed using antibodies directed against RNAPII-S2P (abcam, ab5095). For ChIP-seq, libraries were generated using NEBNext Ultra II DNA Library Prep Kit for Illumina (New England BioLabs) and the final libraries (3 replicates each) were sequenced by the Genomics Core Facility at the University of Regensburg using NextSeq 2000 (Illumina). Reads were aligned to the TAIR10 genome (https://www.arabidopsis.org/) using Bowtie2 (Langmead and Salzberg, 2012 (link)) and coverage tracks were calculated with deeptools “bamCoverage”. Downstream analysis was mainly performed using the deepTools2 suite (version 3.5.0) (Ramírez et al., 2016 (link)) and quality control was performed at several steps using FastQC (Ewels et al., 2016 (link)). Reads were mapped against the TAIR10 genome and regions with aberrant coverage or low sequence complexity were removed (Quadrana et al., 2016 (link)). After confirming high pairwise correlations, the biological replicates were merged and CPM normalized.

ChIP assay was carried out as previously described in ref. ^{39 (link)}, with some modifications. Briefly, we crosslinked control and UVC-treated primary hKCs, lysed cells, and sonicated lysates with the Bioruptor Pico device (Diagenode) at 8 °C for 30 cycles (30 s on, 30 s off). We quantified chromatin with the Qubit system (Thermo Fisher) and incubated 2 ug of crosslinked chromatin with 5 ug of normal rabbit IgG (SCBT, sc-2027) or anti-p53 antibody (rabbit polyclonal, Diagenode C15410083). We used magnetic protein G beads (Dynabeads, Invitrogen 10003D) for immunoprecipitations. Inputs correspond to 1% of the total DNA sample. We de-crosslinked the inputs and immunoprecipitates and purified DNA with MicroChIP DiaPure columns (Diagenode C03040001). We analyzed TINCR p53 RE enrichment over the input chromatin by quantitative real-time PCR (Applied Biosystems) using FastStart Universal SYBR Green (ROCHE) with the following primers: P53 RE TINCR int1 Fw 5′-CAACATGGTGAAACCCCATC-3′, and P53 RE TINCR int1 Rv 5′-CGCCTCCCAGACTCAAG-3′.

One or two-days post infection, cells were collected and washed in PBS. Cells were resuspended in HED buffer (20mM HEPES pH 7.4, 5mM EDTA, 1mM DTT, 10% glycerol) supplemented with cOmplete Protease Inhibitor Cocktail (Roche). Cells were then submitted to one freeze/thaw cycle and sonicated 15 cycles of 30 sec ON / 30 sec OFF using Bioruptor Pico device (Diagenode) at 4°C. Cell lysates were spun down at 14,000 rpm for 15 minutes to remove cell debris. Proteins were quantified using Pierce BCA Protein Assay Kit (ThermoFisher Scientific). Forty μg of proteins were incubated overnight at 37°C with 1 pmol of a fluorescent oligo substrate (5’-ATTATTATTATTCAAATGGATTTATTTATTTATTTATTTATTT-Cy5-3’), 1mM ZnCl₂, 0.025U uracil DNA glycosylase (NEB), 2 μL 10x UDG buffer (NEB) and 100 μg/mL RNAse A (Thermofisher Scientific). Reaction mixture was treated with 50 mM NaOH and heated to 95°C for 10 minutes to cleave DNA probes at the abasic site. Reaction mixture was then neutralized with 50 mM HCl and mixed with 1.25x formamide buffer. Substrates (43 bases-long) were separated from products (30 bases-long) on a 15% tris-borate-EDTA (TBE)-urea gel. The Cy5-labeled substrates and deamination products were detected using ImageQuant LAS4000 mini (GE Healthcare Life Sciences). Densitometry analysis was done using ImageJ software [51 (link)].

1 million mESCs were grown for 24 hours on 100mm dishes and then induced with doxycycline (0.25 ug/mL) for 12 hours. Cells were trypsinized, rinsed once with PBS, and lysis buffer (2 mM Tris-HCl, pH=8.0, 137 mM NaCl, 1% NP-40, 2 mM EDTA, 1x protease inhibitors) was added to lyse cells. Cells then rotated for 30 min at 4°C and were further sonicated for 10 min with a 30-s on-off cycle in a Diagenode Bioruptor Pico device to solubilize chromatin-bound proteins. Protein lysates were quantified with the Bio-Rad (500-0001EDU) Bradford reagent. 3X FLAG Peptide-coupled beads (Sigma Aldrich, F4799-4MG) were pre-washed with lysis buffer, 1 mg of protein was added to 40 μL of pre-washed beads, and samples were incubated at room temperature for 4 hours. Beads were pelleted by low-speed centrifugation. Samples were washed 5X with wash buffer (10 mM TRIS [pH 7.4], 1 mM EDTA, 1 mM EGTA; pH=8.0, 150 mM NaCl, 1% Triton X-100, 0.2 mM sodium orthovanadate, 1x protease inhibitors [see above]), boiled at 95°C in 30 μL 4X Laemelli buffer, and run on an SDS-page gel for western blotting analysis.

Tissue samples from CRC tumors and normal adjacent tissues were homogenized in tissue extraction buffer × 4 v/v (buffer to tissue volume) composed of 60 mM KCl, 15 mM NaCl, 4 mM MgCl2, 15 mM HEPES pH 7.6, 0.5% Triton, 0.5 mM Dithiothreitol (DTT) and 1 tablet (for 20 ml of the buffer) of Roche protease inhibitors (EpiQMAx, GmbH, Planegg, Germany). Homogenization was performed by douncing the tissue 10 × in 1.5 ml reaction tubes, followed by 12 cycles (30 s ON/30 s OFF) of sonication in a Bioruptor Pico device (Diagenode SA, Belgium). Samples were then centrifuged at 13,000 rpm for 30 min at 4 °C. Basic proteins (i.e., histones and others) were extracted as previously described using 0.2 M H₂SO₄ overnight, precipitated with trichloroacetic acid for 2 h, centrifuged at 13.000 rpm for 30 min at 4 °C, and pellets were washed 4 times with 200 µl of ice-cold acetone^{38 (link)}. Pellets were finally resuspended in × 1 (v/v) Laemmli buffer and loaded onto a 4–20% SDS gradient gel (Serva, Germany). Histones bands were cut and processed as described previously for the analysis by mass spectrometry^{39 (link)}.

ChIP was performed utilizing a ChIP-IT High Sensitivity kit (Active Motif; Carlsbad, CA) according to the manufacturer’s instructions. Briefly, 3T3-L1 cultured cells (~80% confluency) were fixed in a formaldehyde-containing buffer that would also fix any DNA-binding protein complexes to the chromatin. These fixed cells were then lysed by repeated snap-freezing cycles and the chromatin sheared via sonication using a Bioruptor Pico device (Diagenode) until the resulting chromatin fragment size was approximately 400-500 bp. Aliquots of recovered sonicated chromatin (15-30 μg) were incubated with 4 μg of Par-4 antibody overnight at 4°C. The antibody-bound chromatin was pulled-down using G-protein agarose beads, the DNA was eluted and subjected to qPCR using the proprietary ChIP-verified negative and positive control primer sets obtained from Active Motif. Remaining DNA was subjected to ChIP-Seq at the University of Kentucky Markey Cancer Center Oncogenomics Shared Resource Facility. Library preparation was performed using the MicroPlex Library Preparation Ki v2 (Diagenode) according to manufacturer’s instructions. Libraries were sequenced on an Illumina HiSeq 2500 using rapid mode run with 50-bp single-reads.
For data analysis, Bowtie2 was used to map the reads. MACS2 was used for peak assignment and peak annotation was done using HOMER.