Igepal ca 630

LNCaP cells were lysed with IGEPAL CA-630 buffer (50 mM Tris-HCl, pH 7.4, (Sigma, T5030), 1% IGEPAL CA-630 (Sigma, I8896), 10 mM EDTA, 150 mM NaCl, 50 mM NaF, 1 μM leupeptin (Sigma, L5793), and 0.1 μM aprotinin (Sigma, SRE0050)). Primary antibody was covalently immobilized on protein A/G agarose using the Pierce Crosslink Immunoprecipitation Kit according to the manufacturer’s instructions (Thermo Scientific, 26147). Samples were incubated with immobilized antibody beads for at least 2 h at 4 °C. Cell lysates were also subjected to immunoprecipitation with either mouse IgG isotype control (Cell Signaling Technology, 5415) or rabbit IgG isotype control (Cell Signaling Technology, 3900), depending on the immunoglobulin type of primary antibody. After immunoprecipitation, the samples were washed with TBS five times. They were then eluted with glycine-HCl (0.1 M, pH 3.5) and the immunoprecipitates were subjected to immunoblotting using specific primary antibodies.

m⁶A-MeRIP using low-input materials was performed based on a previously described protocol [55 (link)] with some modifications. Briefly, total RNA from about 2500 mouse GV oocytes from 5 weeks C57BL/6 J mice (Beijing Vital River Laboratory Animal Technology Co., Ltd) was first randomly fragmented to ~200 nt with RNA fragmentation reagents (AM8740, Thermo, USA), and then incubated with the protein A beads (10001D, Thermo, USA) coupled with anti-m⁶A polyclonal antibody (ABE572, Millipore, USA) in IPP buffer (150 mM NaCl, 10 mM Tris-HCl, pH 7.4, 0.1% NP-40, 0.4 U/μl RNasin). After immunoprecipitation, the RNA reaction mixture was washed twice in 1 ml of IP buffer, twice in 1 ml of low-salt IP buffer (50 mM NaCl, 10 mM Tris-HCl, pH 7.4, 0.1% IGEPAL^® CA-630 (I8896, Sigma-Aldrich, Germany) in nuclease-free H₂O), and twice in 1 ml of high-salt IP buffer (500 mM NaCl, 10 mM Tris-HCl, pH 7.4, 0.1% IGEPAL CA-630 in nuclease-free H₂O) for 5 min each at 4 °C. After extensive washing, the m⁶A-enriched RNA fragments were eluted from the beads by proteinase K digestion followed by phenol-chloroform extraction and ethanol precipitation. The purified RNA was subjected to library construction using the SMARTer Stranded Total RNA-Seq Kit v2 (634413, Clontech, Japan) according to the manufacturer’s instructions. Sequencing was performed on an Illumina HiSeq X-ten sequencing system.

Cells were harvested and nuclear extracts were prepared as previously described37 (link). For extraction of nuclear extracts, cells were harvested and incubated in hypotonic buffer (10 mM Hepes, pH 7.9, 1.5 mM MgCl₂, 10 mM KCl) on ice for 10 min. Cells were transferred to a dounce homogenizer in hypotonic buffer supplemented with 0.1% Igepal CA630 (Sigma) and 0.5 mM DTT and lysed by 40 strokes. Nuclei were washed once in 1 × PBS and extracted in hypertonic buffer (420 mM NaCl, 20 mM Hepes, pH 7.9, 20% glycerol, 2 mM MgCl₂, 0.2 mM EDTA, 0.1% Igepal CA630 (Sigma), 0.5 mM DTT) for 2 h at 4 °C on a rotating wheel.

C-terminally tagged Smc3 strains were grown to 0.1–0.3 OD₆₀₀, arrested in pre-anaphase (nocodazole), pelleted by centrifugation, resuspended in IPH150 (150 mM NaCl, 50 mM TRIS pH 8, 5 mM EDTA, 0.5% IGEPAL-CA 630 (Sigma), 1 mM DTT, 10 mM Sodium Butyrate, Roche protease inhibitor cocktail, and immediately frozen in liquid nitrogen. Cells were mechanically lysed (Bead-beater, BioSpec) and extracts incubated with EZ-View Red Anti-HA affinity matrix (Sigma). Beads were washed with IPH50 buffer (50 mM NaCl, 50 mM TRIS pH 8, 5 mM EDTA, 0.5% IGEPAL-CA 630 (Sigma), 1 mM DTT, 10 mM Sodium Butyrate, Roche protease inhibitor cocktail), and bead-bound proteins harvested using 4X Laemmli loading buffer (Amresco). Acetylation status was determined by Western blot using 1∶5000 dilution of anti-Acetylated Lysine (Calbiochem) and band densities quantified using Photoshop.

Approximately 10⁶ cells were pelleted for 2 min at 3000 RPM and then washed in 150 μl cold PBS. Cells were pelleted again at 3000 RPM for 2 min. The supernatant was then decanted, and the pellet was resuspended in 90 μl ice-cold 0.1% IGEPAL CA-630 (Sigma-Aldrich #I8896). This resuspension was the whole-cell fraction, and a portion (30 μl) of the resuspension was removed and treated with RSB. The remaining resuspension was pelleted at 3000 RPM for 5 min at 4 °C. The supernatant was the cytosolic fraction, and a portion (30 μl) of the resuspension was removed and treated with RSB. The pellet was then washed in 80 μl ice-cold 0.1% IGEPAL CA-630 (Sigma-Aldrich #I8896) and spun again at 3000 RPM for 5 min. The supernatant was discarded, and the pellet was resuspended in 10 μl HED buffer (25 mM Hepes, 15 mM EDTA, 1 mM DTT, 10% glycerol, pH 7.4). This resuspension was the nuclear fraction and was subsequently treated with RSB.

N. benthamiana leaves or Arabidopsis seedlings were ground to a fine powder in liquid nitrogen with sand (Sigma-Aldrich). Proteins were extracted in buffer containing 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10% glycerol, 10 mM DTT, 1 mM NaF, 1 mM Na2MoO4.2H2O, 1% Phosphatase Inhibitor Cocktails 2 and 3 (Sigma-Aldrich), 1% (v/v) P9599 Protease Inhibitor Cocktail (Sigma-Aldrich), 100 μM phenylmethylsulphonyl fluoride and 0.5% (v/v) IGEPAL CA-630 (Sigma-Aldrich). Extracts were incubated 30 min at 4°C and centrifuged for 20 min at 16,000 g at 4°C. Supernatants were incubated for 1–2 h at 4°C with GFP-Trap (ChromoTek) or ANTI-FLAG M2 Affinity Gel (Sigma-Aldrich), and washed 3–4 times with extraction buffer containing 0.1–0.5% (v/v) IGEPAL CA-630 (Sigma-Aldrich). For GFP-Trap immunoprecipitation, beads were boiled in NuPAGE LDS sample buffer (Thermo Scientific) to release proteins. FLAG peptide was used for specific elution of anti-FLAG immunoprecipitated proteins. For immunoprecipitation in Arabidopsis protoplasts, protoplasts were transfected with indicated plasmids, incubated overnight and then treated with H₂O or 1 μM flg22 or elf18 for 15–30 min. Proteins were extracted with extraction buffer (50 mM HEPES [pH 7.5], 150 mM KCl, 1 mM EDTA, 0.5% Trition-X 100, 1 mM DTT, proteinase inhibitor cocktail) at 4°C. Immunoprecipitation was then carried out as described above.

For preparation of nuclear extracts, cells were harvested and incubated in hypotonic buffer (10 mM Hepes, pH 7.9, 1.5 mM MgCl₂, 10 mM KCl) on ice for 10 min. Cells were transferred to a Dounce homogenizer in hypotonic buffer supplemented with 0.1% Igepal CA‐630 (Sigma) and 0.5 mM DTT and subjected to 40 strokes. Nuclei were washed once in 1× PBS and extracted in hypertonic buffer [420 mM NaCl, 20 mM Hepes, pH 7.9, 20% glycerol, 2 mM MgCl₂, 0.2 mM EDTA, 0.1% Igepal CA‐630 (Sigma), 0.5 mM DTT, complete protease inhibitor (Roche)] for 2 h at 4°C on a rotating wheel.