Metagenome: Mangrove Rhizophora: Rhizosphere
The DNA extracted from mangrove rhizosphere soil sample and root entophytic region using total genomic DNA extractions kits as per manufacturer's instructions.   DNA quantity was measured using Qubit 4.0 fluorometer and DNA sample was amplify using 16s primer set and analyzed by gel electrophoresis. The V3-V4 (Product size ~459bp) region of 16s RNA gene was amplified using specific primers. PCR amplified product will be re-amplified using specific V3-F and V4-R primers with overhang adapter via PCR. V3-F =16S Amplicon PCR Forward Primer TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG V4-R =16S Amplicon PCR Reverse Primer GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC. Afterwards, PCR products were purified by using Ampure XP beads. The purified amplicons were subjected to index PCR using Nextera XT indices kit. The resulting indexed amplicons were purified using AmPure XP beads and checked on Agilent tapestation. Libraries were quantified using Qubit HS and qPCR mix pooled together for sequencing. The amplified libraries were analyzed on TapeStation 4150 (Agilent Technologies) using High Sensitivity D1000 ScreenTape as per manufacturer's instructions. The pooled PCR products (library) were loaded on sequencer for cluster generation by hybridization onto the oligonucleotide-coated surface of the flowcell. Immobilized DNA template copies were amplified by bridge amplification to generate clonal DNA clusters. Sequencing was performed using PE300 kit for sequencing of 16S sequencing on Miseq platform.
SUB14887444
RC