title: GSM3141729: wt_L4_ATAC-seq_rep2; Caenorhabditis elegans; ATAC-seq
library_construction_protocol: 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. ATAC-seq was performed essentially as in (Buenrostro et al. 2013). One million nuclei were resuspended in 47.5 ul of tagmentation buffer, incubated for 30 minutes at 37°C with 2.5 ul Tn5 enzyme (Illumina Nextera kit), and then tagmented DNA purified using a MinElute column (Qiagen) and converted into a library using the Nextera kit protocol. Typically, libraries were amplified using 12–16 PCR cycles.
attributes.tag: GEO Accession
attributes.value: GSM3141729