title: GSM5578312: ATACseq_blastula_rep2; Owenia fusiformis; ATAC-seq
library_construction_protocol: RNA-seq: All the fresh samples were snapped freezed by liquid nitrogen, then extracted using the Monarch total RNA Miniprep Kit; ATAC-seq:50,000 cells (~900 blastula, ~500 gastrula, ~300 elongation, ~150 mature larvae and ~40 competent larvae) were washed in 1 ml cold PBS and resuspended in 100  µl cold ATAC lysis buffer 1 (10 mM Tris-HCl pH 7.5, 10 mM NaCl, 3 mM MgCl2, 0.1% (v/v) IGEPAL, 0.1% (v/v) Tween-20, 0.01% (v/v) Digitonin) and used the pestle to homogenize the samples, then and incubated on ice for 3 minutes to lyse cells. The lysis reaction was stopped by adding 1 ml of cold ATAC wash buffer (10 mM Tris-HCl pH 7.5, 10 mM NaCl, 3 mM MgCl2, 0.1% (v/v) Tween-20) and inverting the tube for 3 times to mix. The nuclei were pelleted by centrifugation at 500xg for 10 minutes at 4℃ in a fixed angle centrifuge. RNA-seq: All the libraries were constructed by Oxford genomic center; ATAC-seq:  The supernatant was removed and the nuclei pellet was resuspended in transposition mix containing 5 µl of 10x TMg buffer (100 mM Tris-HCl pH 8.5, 50 mM MgCl2), 5 µl Dimethyl Formamide, 2.5 µl of 100 nM in-house made Tn5 enzyme, 16.5 µl PBS, 0.5 µl of 1% (v/v) digitonin, 0.5 µl of 10% (v/v) Tween-20, and 20 µl H2O. The transposition reaction was incubated at 37°C for 30 minutes in a thermomixer with 1000 rpm mixing. Transposed DNA was cleaned up using the NEB DNA Cleanup kit following manufacturer's instructions. All 10 µl eluted DNA was subjected to 5 cycles of PCR amplification, and 5 µl of this pre-amplified library was used for qPCR to determine the PCR cycles, finally all the libraries were amplified in 7~12 cycles using the NEBNext High-Fidelity 2X PCR Master Mix. RNA-seq and ATAC-seq