GSM8181470: sp_2dpf_2a_scRNAseq; Strongylocentrotus purpuratus; RNA-Seq
For scRNAseq embryos were collected using a 40 μm Nitex mesh filter and spun down at 500 g for 5 min. Sea water was removed and embryos were resuspended in Ca2+ Mg2+-free artificial sea water, then spun down at 500 g for 5 min and resuspended in dissociation buffer (1 M glycine and 0.02 M EDTA in Ca2+ Mg2+-free artificial sea water). Embryos were incubated for 10 min on ice and dissociated gently via pipette aspiration. Dissociated cells were spun down at 700 g for 5 min and washed several times with Ca2+ Mg2+-free artificial sea water. scRNA-seq: Single cell cDNA libraries were prepared using the Chromium Single Cell 3' Reagent Kit, Chemistry v3 and v3.1. scRNA-seq: Libraries were indexed, multiplexed and sequenced on Illumina NextSeq 500 to generate paired end reads. For ATACseq Embryos were collected using a 40 μm Nitex mesh filter and spun down at 500 g for 5 min. For gut tissue sea water was removed and embryos were resuspended in Ca2+ Mg2+-free artificial sea water, then spun down at 500 g for 5 min and resuspended in dissociation buffer (1 M glycine and 0.02 M EDTA in Ca2+ Mg2+-free artificial sea water); embryos were incubated for 10 min on ice and then continuously pipetted up and down with P1000 micropipette to mechanically separate the gut tissue. Separated guts were collected in 100  μl of artificial sea water on ice. The guts or whole embryos were washed in 4 °C artificial sea water, the lysed in 50 μl of 10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.2% IGEPAL CA-630 lysis buffer. Nuclei were centrifuged at 500g, supernatant discarded. 75000 nuclei were tagmented in 2x tagmentation buffer (20 mM Tris-HCl, 10 mM MgCl2, 20% (vol/vol) dimethylformamide), 23.75 μl of nuclease free water and 1.25 μl of Tn5 enzyme from Dr Jose Luis Gómez-Skarmeta. ATAC-seq: The tagmented DNA was purified using MinElute Kit (Qiagen) and eluted in 10 μl of elution buffer, and amplified (10 μl of eluted tagmented DNA, 10 μl of nuclease free water, 2.5 μl 10 μM Nextera Primer 1, 2.5 μl 10 μM Nextera Primer 2.X with following program: 72°C for 5 min, 98°C for 30 sec, then 15 cycles of 98 °C for 10 sec, 63 °C for 30 sec and 72 °C for 1 min, followed by a hold step at 4°C). The resulting library was purified using MinElute Kit (Qiagen) and eluted in 20 μl of elution buffer. ATAC-seq: Indexed libraries were multiplexed and sequenced on Illumina HiSeq 4000 at  BGI Tech (HongKong) to generate paired end reads.