title: scRNAseq of Orbicella faveolata: Bleached
design: A single coral core was randomly selected from the heat-stress treatment tanks and placed into 50 mL Falcon tubes containing 10 mL of calcium-free artificial seawater with 5 mM EDTA (CFASW w/ EDTA) for 1 minute to reduce loosely associated matter from the corals. The cores were then transferred to a fresh 15 mL Falcon tube containing 10 mL CFASW w/ EDTA and left to incubate at room temperature for 5 minutes to allow the coral tissue to loosen from the skeleton and reduce coral mucus. Following this, coral tissue was removed from each coral cores calcium carbonate skeleton using an airbrush (Master Airbrush Cool Runner II Dual Air Fan Air Compressor Professional Airbrushing Kit) with ACME dissociation and cell fixation media (Garca-Castro et al., 2021) and collected into a sterile plastic bag. Once sufficient tissue was removed from the skeleton an additional 4 mL of ACME solution was added to the cell suspension and left to incubate for 1 hour at room temperature on a rocker set to 120 rpm. Every 15 minutes during incubation, a wide-bore pipette was used to mix the cell suspension and further dissociate cells and break-down clumps. After dissociation and fixation, the cell suspension was filtered through a 70 mm MACS SMART-FILTER. The filtered cell suspension was then centrifuged at 450 x g at 4C for 7 minutes, then re-suspended in cold 997 mL 1x PBS + 0.5% BSA and 3 L of RNAse inhibitor. 1 mL 5 mM DRAQ5 and 0.8 mL 14.3 mM DAPI were then added to the cell suspension and allowed to incubate in the dark on ice for 45 minutes. The stained cell suspension was then spun down at 450 x g at 4C for 7 minutes then re-suspended in 2 mL 1x PBS + 0.5% BSA and 2 mL of RNAse inhibitor. The cells were sorted using a SONY SH800S Cell Sorter (Sony, New York, USA) with pre-chilled sample and collection chambers at 5C. Cells were sorted with less than 50 PSI pressure to minimize cell lysis and rupture, first gating for cells with FSC-A values larger than a 1 mm bead marker (Fluoresbrite YG Carboxylate Microspheres-1.00 mm, Polysciences, Pennsylvania, USA), then gating for DAPI+ and DRAQ5+ cells. Collected cells were spun down at 450 x g at 4C for 7 minutes then re-suspended in cold 898 mL 1x PBS + 0.5% BSA and 2 mL of RNAse inhibitor. 100 mL of DMSO was then added to the cell suspension before immediate cryopreservation at -80C until sample submission. For single-cell isolation, 4 cryopreserved cell suspensions representing different staged of coral bleaching were thawed on ice, then spun down at 450 x g at 4C for 7 minutes then re-suspended to a concentration between 700-1,200 cells/mL. The cell suspension was then submitted to the Onco-Genomics Shared Resource at University of Miamis Miller School of Medicine for isolation and sequencing on a 10X Genomics Chromium Controller targeting 10,000 cells with 10X 3 v3.1 chemistry. cDNA libraries were checked using an Agilent 5200 Fragment Analyzer and a Thermofisher Qubit 4 Fluorometer before sequencing on an Illumina NovaSeq 6000 using the NovaSeq S4 200 V1.5 Kit targeting 500,000,000 reads (50,000 reads per cell). Low-quality reads (< 20 Q-score) and trailing poly-T or poly-N tails were trimmed using CutAdapt with Trim Galore (F, 2015). A STAR reference (Dobin et al., 2013) was generated incorporating the genomes of Orbicella faveolate (Young et al., 2024), Breviolum minutum (Shoguchi et al., 2013), Durusdinium trenchii (Shoguchi et al., 2021), Cladocopium goreaui (Shoguchi et al., 2018), and Eimeria tennella (Reid et al., 2014). The trimmed reads were then mapped to this reference using STARsolo (--solotype CB_UMI_Simple --soloMultiMappers EM) (Kaminow et al., 2021) to generate a count matrix. Maximum-likelihood Estimation was used to distribute multi-gene UMIs among their genes while accounting for other UMIs from the same cell as validated by prior studies (Kaminow et al., 2021; Srivastava et al., 2019). The resulting count matrix was imported into R using Seurat for further pre-processing and data visualization (Melsted et al., 2019). Each scRNA-seq data was filtered to remove cells with less than 200 UMIs and cells (or potential doublets) with UMI counts in the top 99% quantile. Each dataset was then Log Normalized with a scale factor of 10,000 before integration into a single dataset using anchor features as determined by Seurats `FindIntegrationAnchors` function. Clusters were identified using the `FindClusters` command using a resolution of 0.3 and algorithm 1. Uniform Manifold Approximation and Projections (UMAPs) of the data into 2-D space was done using 20 dimensions. Cell cluster marker genes were identified using a Wilcoxon Rank Sum test using only genes detected within at least 1% of the cells in a cluster, had a minimum difference of 25% between the other cell clusters, had a log-fold change difference of at least 0.25, and a corrected p-value less than or equal to 0.05. GO analysis was performed using TopGO with only the annotated significant differential expressed genes within at least 1% of the cells in a cluster and a corrected p-value less than or equal to 0.05 mapped to the genome wide annotation for Homo sapiens (org.Hs.eg.db). O. faveolata cell clusters were identified using matching cnidarian cell type marker orthologs between our dataset and the scRNAseq datasets for Hydra vulgaris (Siebert et al., 2019), Nematostella vectensis (Seb-Pedrs et al., 2018), Stylophora pistillata (Levy et al., 2021), and Xenia sp. (Hu et al., 2020) using Broccoli (Derelle et al., 2020). Cell type marker genes from the other cnidarian datasets which were highly expressed in an O. faveolata cell cluster were designated and used as evidence for cell cluster identity. Some O. faveolata cell clusters required additional literature evidence based off the cell cluster marker genes identified above for identification. Symbiont gene expression was compared across Symbiodiniaceae species also using common orthologs identified by Broccoli.
library_name: Ofav_scRNAseq_bleached
identifiers.namespace: SUB14852101
identifiers.id: Ofav_scRNAseq_bleached