title: GSM6744438: 24hr Gastrula Cells, replicate 4, scRNAseq; Nematostella vectensis; RNA-Seq
library_construction_protocol: Gastrula stage embryos were dissociated in calcium- and magnesium-free 1/3X seawater (CMF/NM) with ethylenediaminetetraacetic acid (EDTA) and 0.25% trypsin (henceforth called dissociation solution, pH 7.4-7.6) at 37°C as previously described (Torrez-Mendez 2019), with modifications. Briefly, cells were acclimated into the dissociation solution through a stepwise incubation first in CMF/NM (5 minutes RT, pH 8.0), then with CMF/NM + EDTA (10 minutes RT, pH 8.0), and finally incubated in pre-warmed dissociation solution for ~20 minutes at 37°C, with gentle pipetting every 5 minutes, until a single cell suspension was obtained. Once cells were fully dissociated, an equal volume of cold CMF/NM + 0.05% BSA (pH 7.8) was added. The single-cell suspension was then filtered through a 30µm filter and spun 300 rpm for 15 minutes at 4°C to pellet the cells. Cells were washed and resuspended 3x in cold CMF/NM + 0.05% BSA (spun at 300 rpm for 7 minutes at 4°C). Cell viability was determined by staining the cells with Hoechst 33342 (10mg/µl) and Sytox green for 20 minutes, and quantifications were performed using a hemocytometer. Library construction was performed using the Chromium Next GEM single Cell 3' v3.1 protocol (10x Genomics) following manufacturer's instructions. The concentration of the cell suspension + master mix added to the chip for GEM generation was calculated to target 10,000 cell recovery. Four separate replicates were run on a single chip, and 4 separate cDNA libraries were generated. Quality of the final libraries was assessed using a Bioanalyzer (Agilent). Library was performed according to the manufacter's instructions (single cell 3' v2 protocol, 10x Genomics). Briefly, GCs were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM). The poly-A RNA from the cell lysate contained in every single GEM was retrotranscripted to cDNA, which contains an Ilumina R1 primer sequence, Unique Molecular Identifier (UMI) and the 10x Barcode. The pooled barcoded cDNA was then cleaned up with Silane DynaBeads, amplified by PCR and the apropiated sized fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction Ilumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added.