GSM3142718: wt_L4_DNase-seq_rep1_800U_ml; Caenorhabditis elegans; DNase-Hypersensitivity
Frozen embryos or worms were broken by grinding in a mortar and pestle or smashing using a Biopulverizer, then the frozen powder was thawed in 10 ml Egg buffer (25 mM HEPES pH 7.3, 118 mM NaCl, 48 mM KCl, 2 mM CaCl2, 2 mM MgCl2). Ground worms were pelleted by spinning at 1500 g for 2 minutes, then resuspended in 10ml working Buffer A (0.3M sucrose, 10 mM Tris pH 7.5, 10 mM MgCl2, 1mM DTT, 0.5 mM spermidine 0.15 mM spermine, protease inhibitors (Roche complete, EDTA free) containing 0.025% IGEPAL CA-630. The sample was dounced 10X in a 14ml stainless steel tissue grinder (VWR), then the sample spun 100g for 6 min to pellet large fragments. The supernatant was kept and the pellet resuspended in a further 10 ml Buffer A, then dounced for 25 strokes. This was spun 100g for 6 min to pellet debris and the supernatants, which contain the nuclei, were pooled, spun again at 100g for 6 min to pellet debris, and transferred to a new tube. Nuclei were counted using a hemocytometer. One million nuclei were transferred to a 1.5 ml tube and spun 2000g for 10min to pellet. Replicate concentration courses of DNase I were performed for each stage as follows. One million nuclei were digested for 10 minutes at 25C using 2.5, 5, 10, 25, 50, 100, 200, and 800 units/ml DNase I (Roche), then EDTA was added to stop the reactions. Embryo micrococcal nuclease (MNase) digestion concentration courses for embryos were made by digesting nuclei with 0.025, 0.05, 0.1, 0.25, 0.5, 1, 4, 8, or 16 units/ml MNase in 10mM Tris pH 7.5, 10mM MgCl2, 4mM CaCl2 for 10 minutes at 37C. Reactions were stopped by the additon of EDTA. Following digestions, total DNA was isolated from the nuclei following proteinase K and RNase A digestion, then large fragments removed by binding to Agencourt AMPure XP beads (0.5 volumes). Small double cut fragments < 300 bp were isolated either using a Pippen prep gel (protocol 1) or using Agencourt AMPure XP beads (protocol 2). DNA was converted into sequencing libraries using the Illumina Truseq kit.
GEO Accession
GSM3142718