title: GSM8864601: pl_2wpm_juvenile_J2_snRNAseq; Paracentrotus lividus; RNA-Seq
library_name: GSM8864601
library_construction_protocol: Juveniles were placed in the lid of a 5 mL Protein LoBind tube (Eppendorf, #0030108302) containing 560 μL of pre-chilled Homogenization Buffer. Homogenization Buffer is a hypotonic buffer composed of 250 mM sucrose, 25 mM KCl, 5 mM MgCl2, 10 mM tris buffer (pH 8.0), 1 mM dithiothreitol, 1× protease inhibitor (Sigma-Aldrich, #11873580001), 0.4 U/μL ribonuclease inhibitor (Invitrogen, #AM2682), 0.2 U/μL SUPERase-In ribonuclease inhibitor (Invitrogen, #AM2694), 1 mM spermine, and 0.3% Triton X-100. For each library, the  six juveniles were cut in half using a microsurgical knife (Electron Microscopy Sciences, #72047-30), while the tissues from three of them were further gently scraped using a microsurgical blade and fine forceps (Fine Science Tools, #10316-14 and #11251-20, respectively) under a dissecting microscope. Juvenile large fragments were transferred, using a P1000 micropipette, into a clean 1.5 mL Protein LoBind tube. The rest of the supernatant, was filtered through a 40 μm cell strainer (PluriSelect Mini strainer), to remove debris. Nuclei were extracted through application of mechanical force via pipette aspiration for 10 times using a P1000 micropipette and 15 times using a P200 micropipette. Samples were left until the large juvenile fragments settled and the supernatant containing the isolated nuclei was transferred into a new clean 1.5 mL Protein LoBind tube. Isolated nuclei were spun down for 10 min at 500 g and 4°C using a 5424 R Eppendorf microcentrifuge. The presence of pellets was visually confirmed (brown colored pellets). All the supernatant was discarded except from 50 μL (surrounding the pellet). 600 μL of Wash Buffer [1x Dulbecco's phosphate-buffered saline (DPBS), 2% BSA, 0.2 U/μL SUPERase-In ribonuclease inhibitor, 1 mM spermine and 5 mM MgCl2] was carefully added  using a P200 micropipette, in order not to disrupt the pellet. Next, the nuclei pellets were resuspended through pipette aspiration for 5 times, ensuring that the resuspended nuclei stay close to the bottom of the tube. Isolated nuclei were once more spun down for 10 min at 500 g and 4°C using a 5424 R Eppendorf microcentrifuge. Most of the supernatant was discarded apart from 25 μL (covering the pellet). Lastly, 25 μL of Resuspension Buffer [3x DPBS, 2% BSA, 0.4 U/μL ribonuclease inhibitor, 0.2 U/μL SUPERase-In ribonuclease inhibitor and 1 mM spermine] were added to the pellet. Pellet was resuspended through gentle pipette aspiration for 20-30 times using a P200 micropipette. Nuclei suspensions were filtered through a 10 μm cell strainer (PluriSelect Mini strainer) to avoid aggregates. The nuclei quality and numbers were estimated by Trypan blue and hemocytometer, respectively. Isolate nuclei (25,000 per library) were loaded on the 10x Genomics Chromium Controller according to the manufacturer's instructions. cDNA libraries were prepared using the Chromium Single Cell 3' Reagent Kit (v3.1 Chemistry Dual Index). Libraries were sequenced by the IGFL sequencing platform (Plateforme de Séquençage de l'IGFL) using the Illumina NextSeq 500.
identifiers.namespace: GEO
identifiers.id: GSM8864601_r1