title: GSM8196251: efssdg25, RNAseq, rep1; Arabidopsis thaliana; RNA-Seq
library_construction_protocol: RNA-seq: Total RNA was extracted from 14 days old seedlings using the TRIzol and sent to NGS company for library construction and sequencing 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