title: GSM8631986: efs, acmo1, K36me3_1; Arabidopsis thaliana; ChIP-Seq
library_name: GSM8631986
library_construction_protocol: ChIP for histone modifications was carried out based on both Caroline Dean and Andrzej T. Wierzbicki's protocols (Song et al., 2014) . In brief, 2 g of 14 days seedlings were cross-linked with 1% formaldehyde. The cross-linking was stopped by adding 0.125 M glycine and the sample was rinsed five times with ddH2O. Then the plants were frozen with liquid nitrogen, ground into fine powder, and lysed with Honda buffer (containing 0.44 M Sucrose, 12.5% Ficoll, 2.5% Dextran T40, 20 mM Hepes KOH pH 7.4, 10 mM MgCl2, 0.5% Triton X-100, 1 mM PMSF, 1% Plant protease inhibitor cocktail, 5 mM DTT). The homogenate was filtered through Miracloth (Millipore) and then spun at 2000g for 15 min. The nuclear pellet was washed twice with the same buffer mentioned above and resuspended in lysis buffer (50 mM Tris-HCl, pH 8.0, 10 mM EDTA, 1% SDS, 1 mM PMSF and 1x protease inhibitor) and sonicated using a Bioruptor Pico with the following settings: 30s on/off intervals, four times for 10 min each. The sample was spun at maximum speed for 10 min. The supernatant was collected and diluted 10-fold with ChIP dilution buffer (1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl, pH 8.0, 167 mM NaCl). The diluted chromatin solution was incubated with 5 µg of anti-H3K36me1 (ab9048; Abcam), anti-H3K36me2 (ab9049; Abcam) or anti-H3K36me3 (ab9050; Abcam), anti-H3K27me3 (Millipore, 07-449) at 4 °C overnight.The beads were washed with 1 ml of low-salt wash buffer (50 mM Tris–HCl, pH 7.8, 150 mM NaCl, 1 mM EDTA, 0.1% SDS, 1% Triton X-100) once, with 1 ml of high-salt wash buffer (wash buffer with 500 mM NaCl) twice, with 1 ml of LiCl buffer (10 mM Tris–HCl, pH 7.8, 1 mM EDTA, 0.25 M LiCl, 1% sodium deoxycholate) once and with 1 ml of TE buffer (10 mM Tris–HCl, pH 7.5, 1 mM EDTA) . ChIP-seq: The first adapter was ligated to the 3' end of the ssDNA using Adaptase (Swift Biosciences) via a highly efficient, proprietary reaction that only tails 3' ends of ssDNA and ligates the first truncated adapter to 3' ends simultaneously. This method avoids the bias inherent in random primer-based methods, as it ligates adapters in a sequence-independent manner. The extension step was performed using the primer paired to the first adapter, followed by a ligation reaction to add the second truncated adapter to the 5' ends. An indexing PCR step was performed to add the indexed sequence, and the library was amplified. NET-seq/NET-seq was performed as described (Zhicheng Dong lab, Nature plants-under revision). In brief, 12 days old seedlings were grounded in liquid nitrogen and resuspended in ice-cold lysis buffer (50mM HEPES [PH 7.5], 150mM NaCl, 1mM EDTA [PH 8], 1% triton X-100, 10% glycerol, 5mM β-mercaptoethanol, 1mM PMSF, 2μg/μl Aprotinin and 2μg/μl Pepstatin A). After that lysis mixture was filtered and centrifuged to discard the supernatant. Nuclear pellet was washed once with HBB (25mM Tris-HCL [PH7.6], 0.44M sucrose, 10mM MgCl2, 0.10% triton-100, 10mM β-mercaptoethanol) and HBC (20mM Tris-HCL [PH7.6], 0.352M sucrose, 8mM MgCl2, 0.08% triton-100, 8mM β-mercaptoethanol and 20% glycerol) buffers, respectively, and resuspended in MNase buffer (20mM Tris-HCL [PH 8], 5mM NaCl, and 2.5mM CaCl2) supplemeneted with 20U of MNase. The reaction mixture was incubated at 37C for 5 minutes and the reaction was later stopped with EDTA. After MNase digestion, sonication was performed with 1s ON/OFF for 10 cycles and centrifuged to obtain the supernatant. Then the supernatant was incubated with antibody (Abcam-Ab817) coated beads for 2 hrs at 4C and the beads containing complex was washed 8 times with NET-2 buffer (50mM Tris-HCL [PH7.4], 150mM NaCl, and 0.05% NP-40). Finally, the beads were resuspended with PNK reaction mixture containing 75 ul PNK buffer, 10ul PNK enzymes (NEB) and 15ul of 10mM ATP and incubated at 37C for 10 minutes on a thermomixture (1400rpm). After that supernatant was removed and beads were washed with NET-2 buffer. The beads were again resuspended in 500ul TRIzol and incubated the mixture for 10 minutes at room temperature. After chloroform purification, RNA was precipitated with 1/10 volume of Ammonium acetate, 3 volume of ethanol and 1ul of GlycoBlue. The precipitated RNA was run on 8% PAGE gel and the gel corresponding to 35-100bp size of RNA was sliced. After RNA recovery from the gel, NEXTflex TM small RNA-Seq Kit V3 was used to construct the libraries. Finally, the libraries were recovered from the 6% PAGE gel corresponding to 140-250 bp size.