LchaXBP1 homologs sequencing and quantitation

Total RNA was extracted from tissues of sponge individuals exposed to Present Day control and RCP 8.5 treatments (n=3 per treatment) using TRIzol reagent (Invitrogen). Contaminating DNA was removed using the TURBO DNA-free Kit (Invitrogen). cDNA synthesis was carried out using the GoScript™ Reverse Transcriptase kit (Promega).Primers were designed to target the full-length and the bZIP domain of the two XBP1 homologs, as well as the β-tubulin gene (endogenous control for qPCR) in L. chagosensis.Full-length and bZIP domain of LcXBP1 genes were amplified from the prepared cDNA using the following primers: LcXBP1_1 (F: 5’-GTTCGTGTTGTAGTTGTAGAGTAG-3’; R: 5’- TTATTTGGCCGCATTCATTTACAC -3’), LcXBP1_2 (F: 5’-CATTAGCCAGAGCGTTGGATTGTC-3’; R: 5’-AGATCCCTTCATCCTCTTCCGAATTC-3’), LcbZIP2650 (F: 5’-CGAGAAGCTTGACCACCTGT-3’; R: 5’-TCTGTCTTACCCCTCCTTGC-3’), and LcbZIP60042 (F:5’-CCTGAAAAGTATCGGCCGAG-3’; R: 5’-GTGAGGCTGGCAATGTTGAA-3’). Amplified samples were viewed through agarose gel electrophoresis to confirm amplicon size and were sent to Macrogen, South Korea for Sanger sequencing. Trimmed and aligned sequences were deposited to Genbank under the accession numbers: PP716769 (LcXBP1_1), PP627506 (LcXBP1_2), PP627507 (LcbZIP2650), and PP627508 (LcbZIP60042).Expression levels of the two XBP1 homologs in L. chagosensis under the Present Day and RCP 8.5 treatments were quantified using the bZIP primers through QuantStudio™ 3 Real-Time PCR system (Thermo Fisher Scientific). qPCR reactions included 40 cycles of activation at 95°C for 2 min, denaturation at 95°C for 15 sec, and annealing/elongation at 55°C for 1 min. Each reaction contained 10 μl of 2 × GoTaq® qPCR Master Mix (Promega), 0.2 μl each of 100 μM forward and reverse primers, 4 μl template cDNA, and 5.6 μl nuclease-free H2O. Three biological replicates and three technical replicates were used in the quantitation of each gene alongside negative controls. Primer efficiency and primer specificity were assessed using the dilution curves and melt curves, respectively. The abundance of target transcripts was computed using the ddCt and Pfaffl methods. Target transcript abundances were normalized to β-tubulin as a reference gene.