M8823

Chromatin immunoprecipitation (ChIP) was performed as previously described [52] (link). In brief, 100 ml of exponentially growing yeast cultures were arrested with 20 µg/ml nocodazole for 3 h at room temperature and subsequently cross-linked with formaldehyde at a final concentration of 1%. The cross-linking reaction was stopped with glycine; the cells were harvested and lysed using Silica beads. Chromatin was sheared to 300–500 bp fragments by water-bath sonification (Bioruptor UCD-200, Diagenode). FLAG-tagged proteins were immunoprecipitated using the monoclonal ANTI-FLAG antibody coupled to superparamagnetic beads (Sigma-Aldrich, M8823). DNA was recovered by phenol/chloroform extraction followed by ethanol precipitation. Quantitative RT-PCR was performed using the Light Cycler LC480 system (Roche) to evaluate the enrichment of analyzed proteins. The ratio of DNA-IP to DNA-Input was calculated for centromeric/pericentromeric regions (0.1 kb away from CEN1, 1.1 kb away from CEN4 and 5 kb away from CEN12) as well as for the rDNA locus (NTS1-2). The relative enrichment was calculated by normalization to the IP/Input ratio for a control locus on the arm of chromosome 10. Three independent immunoprecipitation experiments from metaphase-arrested cells were performed for FLAG-tagged Smc2 and Ipl1; Rts1 and Mcd1 ChIP experiments were performed twice.

Tet-on HeLa cells expressing eGFP fusion proteins were generated as described elsewhere (Castello et al., 2012 (link)). Upon induction, cells were UV irradiated and subjected to small scale RNA interactome capture (Castello et al., 2013b (link)). Eluates were measured in a plate reader. For PNK assays, cell monolayers were irradiated with 150 mJ/cm2 UV₂₅₄ (Castello et al., 2013b (link)). After cell lysis and RNase treatment, FLAG-HA tagged proteins were immunoprecipitated with an anti-FLAG antibody coupled to magnetic beads (M8823, Sigma Aldrich) and processed as in Beckmann et al. (2015) (link). More detailed information can be found in the Supplemental Information.

For worm samples, 30,000 young adult worms with the indicated genotype were washed off by M9 buffer and resuspended in 3 ml lysis buffer (50 mM tris-HCl pH 8.0, 137 mM NaCl, 1% Triton X-100, 1 mM EDTA, 10% glycerol, proteinase inhibitor). Samples were homogenized with a glass homogenizer and sonicated. For HEK293T cells, cells in one 10 cm dish with 90% confluence were washed with 1X PBS buffer, then resuspended in 1 ml lysis buffer and sat on ice for 30 min. The worm or cell lysate was centrifuged at 20,000 × g for 15 min. The supernatant was then transferred into a new tube and rotated at 4 °C overnight in the presence of the designated antibody anti-GFP antibody (Abcam #ab290, 1 µl per sample), anti-FLAG magnetic beads (Sigma #M8823, 40 µl per sample), anti-Myc magnetic beads (Bimake #B26302, 20 µl per sample). For anti-GFP immunoprecipitation, protein G beads (Invitrogen #10004D, 40 µl per sample) were subsequently added to each sample and rotated at 4 °C for additional 2 h. After binding, the beads were washed three times with lysis buffer and boiled in 50 µl 2X SDS Laemmli buffer (4% SDS, 20% glycerol, 10% 2-mercaptoethanol, 0.02% bromophenol blue, 0.125 M Tri-HCL, pH 6.8) at 95 °C for 10 min.

For preparation of the protein samples from mycelium, conidia (5 × 10⁵ conidia/ml) of wild type and BagA::Flag strains were inoculated in 50 ml of liquid minimal media and incubated for 24 h at 37°C. Mycelial samples were collected, freeze dried and stored at −80°C. Prepared mycelial samples were resuspended in lysis buffer B250 (100 mM Tris-HCl, pH 7.5, 250 mM NaCl, 10% glycerol, 1 mM EDTA, and one Roche Complete Protease inhibitor tablet without EDTA per 50 ml) as previously described (Park et al., 2012 (link); Patananan et al., 2013 (link)), broken by a mini-bead beater for 2 cycles (1 min homogenization with 10 min sitting on ice) and centrifuged in a microcentrifuge for 15 min at 15,000 rpm at 4°C. The supernatant was incubated with anti-FLAG M2 magnetic beads and elution was performed according to manufacturer's protocol (M8823, Sigma). Total elution fractions were sent for sequencing and further processing.

A P-element based transgenic fly line with an insulated FLAG-BEAF-32B-EGFP gene expressed from its endogenous promoter (Avva and Hart 2016 (link)) on the X chromosome was used to collect 4- to 20-h embryos. These flies also had the wild-type BEAF gene. Control embryos were collected from y¹ w^67c23 flies. Nuclear extracts were prepared as previously described (Zhao et al. 1995 (link)) and immunoprecipitations were done using anti-FLAG M2 coupled to magnetic beads according to the manufacturer’s protocol (Sigma-Aldrich M8823). Two experimental and 3 control coimmunoprecipitation samples passed quality control (SDS-PAGE followed by Western blot for BEAF and silver stain for total protein) and were sent to the Thermo Fisher Scientific Center for Multiplexed Proteomics at Harvard Medical School for tandem mass spectrometry analysis. Differences between the experimental and control samples were evaluated for statistical significance by the Student’s t-test and corrected for multiple testing using the Benjamini–Hochberg correction. Results are in Supplementary Table 1.

The FLS were lysed in an immunoprecipitation lysis buffer containing a protease inhibitor. After centrifugation, the collected supernatant was incubated with magnetic beads (Sigma, M8823) at 4°C overnight. Then, the beads were rinsed, and the bound proteins were eluted and then evaluated using a Western blot assay.