Igg agarose beads

ChIP experiments were performed as described previously^{71 (link)}, with some modifications. Briefly, 150 ml cell cultures were grown to OD₆₀₀ = 0.8–1.0, cross-linked (1% formaldehyde, 16 min, room temperature), and lysed by bead beating. The chromatin fraction was isolated and sheared to 200–500 bp fragments (30 min, 30 s on/off cycles, 90% amplitude) using a Q800R1 sonicator (QSonica). Immunoprecipitations were performed overnight at 4 °C with 100 µl of 25% slurry of prewashed IgG-agarose beads (Sigma) from lysates corresponding to 80–100 OD₆₀₀ of cells. The beads were washed and eluted, and the eluate was reverse cross-linked at 65 °C for 3 h and incubated with proteinase K for 2 h at 55 °C. DNA was cleaned up with ChIP DNA Clean & Concentrator^TM kit (#D5201, Zymo Research). Immunoprecipitated DNA was quantified by qPCR using primaQUANT SYBR Master mix (Steinbrenner Laborsysteme GmbH. #SL-9902B) and a QuantStudio^TM 3/5 Real-Time PCR system (Applied Biosystems/Thermo Fisher). Primers are listed in Supplementary Table 3. To avoid changes due to different rDNA copy number, the relative fold enrichment for each strain was determined over the average of three rDNA positions (RDN1 #9, RDN1 #12, and RDN1 #25), after normalization to input: [rDNA(IP)/rDNA(WCE)]/[mean(rDNA)(IP)/mean(rDNA)(WCE)]. Graphs were generated using Graphpad Prism and Inkscape.

All ChIP-seq experiments were performed in at least duplicates. Chromatin was prepared as previously described (Wittmann et al., 2017 (link)). For spike-in control, S. cerevisiae cells were added to S. pombe cells before cross-linking. S. cerevisiae cells correspond to 15% of S. pombe cells. Immunoprecipitations were conducted with IgG agarose beads (Sigma) or antibodies against Rpb1 (8WG16, Millipore), Tyr1P-CTD (3D12, ActiveMotif), Ser2P-CTD (3E10, Millipore), Thr4P-CTD (6D7, ActiveMotif) coupled to protein G dynabeads (Life Technologies) as described in (Wittmann et al., 2017 (link)) except that when immunoprecipitating with Thr4P antibody, NaCl was used at concentration of 150 mM in all buffers. Libraries were constructed using NEBNext Fast DNA Library Prep Set for Ion TorrentTM Kit or NEBNext Ultra DNA Library Prep Kit for Illumina. Libraries were sequenced on the Ion Torrent Proton or the Illumina NextSeq500.

In order to identify protein phosphorylation at the C-terminal serine cluster of AtRGS1, seven-day-old rgs1-2 seedlings overexpressing TAP-tagged AtRGS1 were treated with 100 nM flg22 for 0, 3, and 15 min. Total protein was extracted as described by (15 (link)). AtRGS1 was purified using IgG-agarose beads (Sigma), and the protein A portion of TAP tag was then partially removed by TEV digestion. Phosphorylation levels at the serine cluster were determined using an anti-phospho-AtRGS1 antibody, which recognizes the pSer428, pSer435, and/or pSer436 with low avidity (10 (link)). Total AtRGS1 levels were determined using an anti-AtRGS1 antibody (9272). Both sera were produced at the University of North Carolina and are available from AgriSera AB. Detection of the phosphorylated AtRGS1 protein in vivo in the atbα mutant or in in vitro in enriched samples was not possible due to the greatly reduced abundance of AtRGS1 protein.
For the stability assay, 7-day-old seedlings were treated with the translation inhibitor cycloheximide at 200 μM for 1 h. Protein levels were compared by Ponceau S staining and AtRGS1 levels were determined by probing with anti-GFP Tag polyclonal antibody (Invitrogen #A-11122). Bands were quantified using the software ImageJ (https://imagej.net/ij/) and the RuBisCO large chain (rbcL) was used as an endogenous control of total protein levels.

Cells were collected in ice, washed with cold lysis buffer, flash-frozen in liquid nitrogen and stored at −80°C. RNA immunoprecipitation (RIP) was done according to the protocol in [28 (link)] with the following modifications. All steps were performed in a cold room on ice. Protease inhibitors leupeptin, pepstatin, bestatin and aprotinin were added to a final concentration of 2 µg ml⁻¹ of lysis buffer. PMSF was added to a final concentration of 1 mM and 100 U ml⁻¹ of SuperaseIn (Ambion) was also added to lysis buffer. Cells were lysed by bead beating using 0.7 mm Zirconia beads in a Fast-Prep at maximum speed for 2 pulses of 20 s and incubation for 5 min on ice in between pulses. 20 µl of lysate was frozen in liquid nitrogen for western, and 50 µl was stored in liquid nitrogen as input. IgG-Agarose beads (Sigma) were used for immunoprecipitation at 4°C for 2 h. Beads were washed for a total of nine washes, three times in lysis buffer, and six times in wash buffer containing NP-40, glycerol, heparin, RNase and protease inhibitors. RNA was eluted from the beads using 1% SDS in 1X TE with SuperaseIn. RNA was purified by phenol:chloroform extraction and sodium acetate and ethanol precipitation.

Cell lysates were prepared by resuspending the pellets from 150 to 200 OD₆₀₀ units in 800 μl lysis buffer (50 mM Tris pH 7.5, 150 mM NaCl, 10% glycerol, 1 mM EDTA pH 8, 0.5% NP-40, 1x complete EDTA-free protease inhibitor cocktail (Roche), 2 mM PMSF, 20 mM N-ethylmaleimide). To block phosphatase activity, 1x tablet of PhosSTOP, 10 mM NaF and 20 mM ß-glycerophosphate were added to the lysis buffer. Cells were lysed by bead beating (Precellys 24, Bertin instruments) with zirconia/silica beads (BioSpec Inc.) and lysates were cleared by centrifugation (800 g, 5 min). Clarified extracts were incubated with pre-equilibrated antiHA-sepharose (Roche), GFP-Trap (Chromotek) or IgG-agarose beads (Sigma) for 1.5 h at 4 °C, beads were washed four times with lysis buffer and two times with wash buffer (50 mM Tris pH 7.5, 150 mM NaCl, 1 mM EDTA pH 8). Proteins were eluted by boiling with 30 µl HU loading buffer and analyzed by immunoblotting.

Nuclear supernatants enriched in supraspliceosomes were prepared as previously described [38 (link),43 (link)] (see also above). The affinity purification was performed as described [41 (link)] with some changes as previously described [34 (link)], and without using the tobramycin step. All steps were conducted at 4°C, with mild agitation. To 500 μl of the nuclear supernatants, originated from ~0.5–1*10⁸ cells, 1 μg of ZZTEVPP7CP protein was added in a solution of: 10% (v/v) glycerol, 0.1% (v/v) NP-40, 0.5 mM phenylmethylsulfonyl fluoride (PMSF), final concentration; and incubated for 90 min with rotation. Next, 250 μg of IgG agarose beads (Sigma Aldrich) pre-washed with binding buffer [10% (v/v) glycerol, 10 mM Tris pH 8, 2 mM MgCl₂, 100 mM NaCl, 1 mM DL-Dithiothreitol (DTT), 0.1% (v/v) NP-40, 0.5 mM PMSF and 2 mM vanadyl ribonucleoside (VR)] were added, followed by additional rotation for 90 min. Three washing steps with binding buffer and another one with TEV buffer (10% (v/v) glycerol, 10 mM Tris pH 8, 2 mM MgCl₂, 100 mM NaCl and 1 mM DTT) were performed for 10 min each. Elution of the bound material from the beads was performed by incubating the beads overnight with 1 μg of TEV protease in 50 μl TEV buffer. The supernatant was then transferred to a new tube. Samples were taken either for protein analysis by WB and for RNA analysis by RT-PCR.