GSM5227621: WT_seedling_Hi-C; Arabidopsis thaliana; Hi-C
Ten-day-old seedlings were harvested and fixed with 20 ml 2% formaldehyde solution for 15 min in vacuum conditions at room temperature and then quenched by adding 2.162 ml 2.5 M glycine. Fixed seedling tissue was rinsed with water three times and dried with tissue paper.   The nuclei were released by grinding in liquid nitrogen and then resuspended with 25 ml of extraction buffer I (0.4 M sucrose, 10 mM Tris HCl pH 8, 10 mM MgCl2, 5 mM β-mercaptoethanol, 0.1 mM PMSF, 13 μl protease inhibitor). Nuclei were filtered through miracloth (Calbiochem) and then centrifuged at 4000 rpm for 20 min at 4 °C. The supernatant was discarded whilst the pellet was resuspended with 1 ml of extraction buffer II (0.25 M sucrose, 10 mM Tris HCl pH 8, 10 mM MgCl2, 1% Triton X-100, 5mM β-mercaptoethanol, 0.1mM PMSF, 13 μl protease inhibitor). Then, the mixture was centrifuged at 14000 rpm for 10 min at 4 °C and the pellet was resuspended with 300 μl of extraction buffer III (1.7 M sucrose, 10 mM Tris HCl pH 8, 0.15% Triton X-100, 2 mM MgCl2, 5 mM β-mercaptoethanol, 0.1 mM PMSF, 1 μl protease inhibitor), load the mixture onto the top of an equal amount of clean extraction buffer III, then centrifuge at 14000 rpm for 10 min. The Hi-C library construction and sequencing were conducted by Annoroad Gene Technology Co., Ltd (Beijing, China). Briefly, The pelleted nuclei were washed twice with 1x ice cold CutSmart buffer and finally resuspended in 0.5 ml volume. SDS was applied to permeabilize nuclei at 65 °C for 10 min, Triton X-100 was added to quench SDS. Thereafter, chromatin was digested with 400 units MboI overnight at 37 °C with gentle rocking. MboI was then denatured to cease activity. Digested chromatin underwent DNA end repair with biotin-14-dCTP insertion followed by blunt-end ligation. After decrosslinking with proteinase K at 65 °C, DNA was purified by phenol chloroform extraction method. Biotin-14-dCTP was removed from non-ligated DNA fragment ends using T4 DNA polymerase. DNA was sheared to a range of 200 to 600 bp by sonication. Next, the fragments underwent end repair and were pulled down by streptavidin C1 magnetic beads to enrich for fragments containing contact information. Fragment ends were then A-tailed, sequencing adapters were ligated, and libraries were amplified by PCR for 12-14 cycles. Following purification, libraries were sequenced using the Illumina HiSeq platform with 2×150 bp length.