title: GSM4293375: N2_ARC_pooled; Caenorhabditis elegans; OTHER
library_construction_protocol: Frozen worm pellets were ground into a fine powder in which worms were broken into ~10 fragments. 1 ml of worm powder was then fixed in 10 ml of 1% formaldehyde in PBS at room temperature (RT) for 10 min with gentle shaking then quenched for 5 min with a final concentration of 125mM glycine. Fixed worm fragments were then washed with Buffer A (340mM sucrose, 15mM Tris-HCl pH7.5, 2mM MgCl2, 0.5mM spermidine, 0.15 mM spermine, 1mM DTT, protease inhibitors), resuspended in 7 ml Buffer A, then the material dounced 20 strokes in a 7 ml stainless steel tissue grinder (VWR-#432-5005). The material was spun at 100g for 5 minutes and the supernatant containing nuclei was transferred to a new tube and kept. The pellet was resuspended in Buffer A and again dounced with 20 strokes. After spinning, the two supernatants containing nuclei were pooled. Three aliquots of 10 million fixed nuclei were spun down at 1,000g and resuspended in 200ul of 1x DNase buffer (Roche) and chromatin was digested with 25, 50, or 100U/ml DNase I at 25C for 10min. The reactions were then quenched with a final concentration of 25mM EDTA and 5mM Tris pH 7.5. Nuclei were washed twice with 1ml of ice-cold Nuclear Washing Buffer (340mM sucrose, 15mM Tris-HCl pH7.5, 25mM EDTA, 0.5mM spermidine, 0.15mM spermine, 1mM DTT, protease inhibitors). Nuclei were then resuspended in 100ul of end repair master-mix (10ul 10x NEB End Repair Buffer, 5ul NEB End Repair Enzyme Mix, 85ul H2O) and incubated at 20C for 30min with rotation. Thereafter, 400ul of ligation master-mix was added (40ul 10x ligation buffer, 5ul T4 DNA ligase [400,000 units/ml], 355ul H2O) and the mixture was incubated at 4C overnight with rotation. Nuclei were pelleted, resuspended in 50ul of tagmentation master-mix (22.5ul H2O, 25ul 2x Nextera TD buffer, 2.5ul Nextera Tn5 transposase) and incubated at 37C for 30 min. 5ul of 1% SDS and 2ul of NEB Proteinase K [800U/ml] was added and left at 65C for 15 min before DNA from the mixture was purified using Qiagen MinElute columns. Large fragments (>500bp) were removed from the purified DNA using two rounds of 0.6vol AMPure XP beads. Prior to PCR amplification, a test qPCR amplification using 1/20th of the input DNA was performed and the cycle number for PCR was determined by the midpoint of the exponential phase of the amplification curve. The resultant DNA was amplified with NEBNext Ultra II Q5 Master-mix under the following PCR conditions: 72 °C for 5 min, 98 °C for 30 s, and cycling at 98°C for 10s, 63°C for 30s and 72°C for 1min using the determined cycle number. The library was size-selected with AMPure XP beads to a final range of 200-700bp (insert size: ~70-570bp). Libraries made from nuclei digested with different concentrations of DNase I were quality tested by qPCR at the gap-3 promoter. DNAse I over- or under-digestion led to poor enrichment of regulatory elements. Good libraries generally had gap-3 promoter enrichment values of >4-fold.
attributes.tag: GEO Accession
attributes.value: GSM4293375