GSM7508881: Zebrafish_WT2; Danio rerio; RNA-Seq
Culture WT, ΔMMAR_0267 and ΔMMAR_0267:: MMAR_0267 M. marinum on 7H10 plates, pick a single colony and culture it in liquid medium. Shake the culture at 30°C until the OD600 value reaches approximately 0.5. Select healthy adult zebrafish from a population and acclimate them in fresh water for 24 hours before grouping. Centrifuge the M. marinum culture, remove the supernatant, wash the bacteria with PBS three times, and finally dilute the bacterial suspension to 10^8 CFU/mL. Place the zebrafish into a new dish, inject 10μL of bacterial suspension into the zebrafish abdomen. After infection, return the zebrafish to the acclimation water. At different time points after infection, observe the fish for signs of illness and death. Simultaneously, isolate and identify the pathogenic bacteria from dead fish to determine the species and quantity of the infecting pathogen. After the samples pass the test, use magnetic beads with Oligo (dT) to enrich Eukaryote mRNA. Subsequently, a fragmentation buffer was added to randomly interrupt the mRNA. Using mRNA as a template, the first strand of cDNA was synthesized using six base random primers. Then, buffer, dNTPs, and DNA polymerase I were added to synthesize the second strand of cDNA. The double stranded cDNA was purified using AMPure XP beads. The purified double stranded cDNA was subjected to end repair, A-tail addition, and sequencing connections. Then, AMPure XP beads were used for fragment size selection, and PCR enrichment was performed to obtain the final cDNA library.