I want to perform PacBio RNA-seq of a filamentous fungal isolate during multiple infection time-points in barley leaf. I was told that PacBio RNA-seq would not be viable due to the low concentration of fungal reads vs. barley reads. It was suggested that if I could purify or separate the fungal tissue from the plant tissue prior to RNA extraction, I would then be able to proceed with sequencing. I have done Illumina mRNA sequencing but for better annotation I would like to do long-read RNA sequencing. Thanks!
Ganesh Saravanan Kathan
- Collection of Leaf Tissue: Select leaves showing visible fungal growth. It’s important to handle leaves carefully to avoid contamination.
- Surface Sterilization: Submerge the leaves in 70% ethanol for 1-2 minutes to sterilize the surface. Follow this with a rinse in sterile distilled water or PBS to remove any residual ethanol.
- Isolation of Fungal Structures:Using sterile forceps and scalpels, carefully cut out small pieces (about 1 cm²) of leaf tissue that show fungal growth. Transfer these pieces onto sterile petri dishes under sterile conditions (preferably in a laminar flow hood).
- Inoculation onto Agar Plates:Prepare sterile culture plates with appropriate growth media (e.g., Potato Dextrose Agar or Sabouraud Dextrose Agar). Place the leaf tissue pieces (containing fungal growth) onto the agar surface, ensuring they are in contact with the media.
- Incubation: Seal the petri dishes with Parafilm and incubate them at an appropriate temperature for fungal growth (typically around 25-30°C, depending on the fungi).
- Observation and Subculturing:Check the plates regularly (every 1-2 days) for fungal growth. Filamentous fungi usually appear as fuzzy or cottony colonies. Once pure colonies of fungi develop, transfer them to fresh plates with the same media to obtain pure cultures. This may involve subculturing by transferring a small piece of fungal mycelium from the edge of a colony to a new plate.
- Storage: Store pure cultures for long-term use by periodically transferring them to fresh media plates or storing them in glycerol suspensions at -80°C.
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