GSM6503215: mature rudiment 1, scRNAseq; Paracentrotus lividus; RNA-Seq
Per biological replicate, three rudiments were collected through dissection using tungsten needles under a stereoscope. Each rudiment was dissected out of the larva, while larval tissues were discarded. Dissected rudiments were dissociated t 37°C for 40 min with a pipette in Ca2+Mg2+ free Artificial Sea Water (CaMg-free ASW) with 1% Protease XIV and 0.05% Liberase. Larval debri were removed with 0.22 µm mesh, the dissociated cells were spun down at 700 g for 5 min and washed several times with CaMg-free ASW. Cell viability was assessed using Propidium Iodide (1µg/mL) and Calcein AM (2 µg/mL). Single cells were counted using a hemocytometer and diluted following 10x Genomix guidelines. Single cell cDNA libraries were prepared using the Chromium Single Cell 3' Reagent Kit, Chemistry v3. Libraries were indexed, multiplexed and sequenced on Illumina NextSeq 500 to generate paired end reads.