GSM5941848: WT_seedling_Hi-C_rep2; 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. Libraries were prepared using the Ovation Ultralow System V2 (Nugen, 0344) and sequenced on the NextSeq 500 (Illumina) with 76 bp single end reads at the John Innes Centre, Norwich, UK.