Coral Rubble Metagenome
Coral Rubble samples preserved in DESS solution (at 1:3 ratio and stored at 4C until processing) were used for extracting high molecular weight DNA under aseptic conditions using the Fast DNA Spin Kit for soil (MP Biomedicals, USA) as per the manufacturers protocol. DNA quality was assessed via agarose gel electrophoresis, NanoDrop ND-2000 (Thermo Fisher, USA) for purity, and Qubit 3.0 Fluorometer (Invitrogen, USA) for quantification. 16S rRNA gene amplification (V3-V4 region) was performed using universal primers 338F and 806R. PCR conditions: initial denaturation (95C, 2 min), 30 cycles of denaturation (95C, 20s), annealing (55C, 30s), extension (72C, 20s), and final extension (72C, 5 min). Amplicons (~380-400 bp) were confirmed by 2% agarose gel electrophoresis and purified using bead purification. For adaptor ligation, a second PCR was performed with GoldStart Master Mix and UAP1 primers, followed by bead purification. The final library was quantified using Qubit 3.0 and stored at 4C or -20C. For each of the sediment samples, replicate samples were subjected to library preparation and sequencing.
SUB15698175
RUB_C_02