Nematostella vectensis ATAC-Seq sample 1 run 1
Embryos were raised at 21C until gastrula stage (24 hpf). Ten embryos were collected and washed with cold PBS, then cold lysis buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1 % NP-40, 0.1% Tween-20, 0.01% Digitonin) was added. Embryos were disintegrated by pipetting and incubated in lysis buffer for 6.5 minutes on ice. Nuclei were then pelleted by centrifugation (0.5 rcf, 10 min, 4C). The pellet was resuspended in 50 l tagmentation mix (5x TD buffer, 0.1% Tween-20, 0.01% Digitonin, 2.5 l Tn5 transposase). TD buffer and Tn5 transposase were kindly provided by the David Garfield lab. The transposition reaction was incubated at 37C for 30 min with agitation at 1000 rpm and then cooled down on ice for 5 minutes. DNA purification was carried out with the QIAquick PCR kit (QIAGEN, #28104) according to the manufacturers instructions, with an elution in 20 l elution buffer. PCR amplification was performed for 12 cycles according to Buenrostro 2015. A final purification step was performed with the QIAquick PCR kit (QIAGEN, #28104), elution in 15 l EB. The quality of the library was validated with an agarose gel, from which DNA was extracted with a peqlab gel extraction kit, eluted in 20 l EB, and stored at -20C.