title: Bulk RNA sequencing of Orbicella faveolata: Control 3
design: A single coral core was randomly selected from the non-heat-stress (control) 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. These cryopreserved cell suspensions for control samples 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 in Trizol for bulk RNA extraction. Cells in Trizol were then vortexed thoroughly for 10 minutes. A chloroform precipitation was then performed, and linear poly acrylamide was added to pellet the RNA. Following an alcohol wash, extracted RNA was re-suspended in nuclease-free water. 100 x 100 paired-end cDNA libraries targeting 100 M reads per sample were prepped and sequenced at the Onco-Genomics Shared Resource at University of Miamis Miller School of Medicine on an Illumina NovaSeq 6000 using the NovaSeq S4 200 V1.5 Kit. Raw reads were trimmed of low-quality reads (<20 Q-score) and adapters using CutAdapt with Trim Galore. Trimmed reads were then mapped to the same STAR reference generated above for the scRNAseq treatment samples. The count tables were then imported into R and normalized based on library size. A students T-test with a false discovery rate adjustment was used to determine if genes were significant between control samples.
library_name: Ofav_BulkRNA_C3
identifiers.namespace: SUB14852101
identifiers.id: Ofav_BulkRNA_C3