title: GSM4310278: WT_ChIP; Arabidopsis thaliana; ChIP-Seq
library_construction_protocol: Briefly, tissues were cross-linked using 1% formaldehyde and quenched with glycine. To isolate the nuclei or chromatin, approximately 0.5 g of samples were ground in liquid nitrogen into fine powder and resuspended in 30 ml of EB1 buffer (0.4 M sucrose, 10 mM Tris–HCl, 5 mM b-mercaptoethanol, and 1 mM PMSF). The mixture was filtered through Miracloth (Millipore, cat. No. 475855-1R), and the filtrate was centrifuged at 1,800 × g for 10 min at 4 °C. The pellet was washed three times in 1.5 ml of EB2 buffer (0.25 M sucrose, 10 mM Tris–HCl, 5 mM b-mercaptoethanol, 10 mM MgCl2, 1% Triton X-100, and 1 mM PMSF) and centrifuged at 2,000 × g for 10 min at 4 °C. Next, the pellet was washed in 0.5 ml of EB3 buffer (1.7 M sucrose, 10 mM Tris–HCl, 5 mM b-mercaptoethanol, 2 mM MgCl2, 0.15% Triton X-100, and 1 mM PMSF) and centrifuged at 2,000 × g for 1 h at 4 °C. The final pellet was resuspended in 0.25 ml of NLB buffer, and the chromatin was fragmented into 200–500 bp by sonication in a Bioruptor (Diagenode). The chromatin sample was then centrifuged at 2,000 × g for 10 min at 4 °C, and the supernatant was used for IP. ChIP was performed using anti-GFP antibodies (ChromoTek, gtma-20). After washing the bead-IP complexes, ChIP DNA purification, library preparation was prepared using an NEBNext® Ultra™ II DNA library prep kit for Illumina® sequencing (New England BioLabs, E7645). Finally, the DNA library was sequenced using an Illumina HiSeq X Ten system.
attributes.tag: GEO Accession
attributes.value: GSM4310278