My supervisor has already agreed to help a collaborator to sequence total RNA from a pool of plant and bacteria. They want me to do one library set for the plant mRNA (easy enough, capture poly-A mRNA on poly-dT beads), and another library set for the bacterial mRNA. Here are the two options I've come up with for the second bacterial set. I'm hoping someone out there has experience with something like this, and can either give me pointers on these options, or else suggest another option that I haven't thought of. BTW, there are going to be a LOT of samples (time points, triplicates, different temperatures...)
1. Bacterial mRNA can't be pulled out of a mixed pool, so we'd first have to pull out the poly-A plant mRNA (using poly-dT beads from Illumina kit), then pull out the plant rRNA (and plastid RNA) using either RiboMinus Plant kit (Life Technologies, $562 / 8 preps) or RiboZero rRNA Removal kit-Plant Seed/Root (Epicentre, $554 / 6 preps), then pull out the bacterial rRNA using either RiboMinus Transcriptome Isolation Kit - bacteria (Life Technologies, $493 / 12 preps), MICROBExpress bacterial mRNA enrichment kit (Life Technologies, $420 / 20 preps), or RiboZero Magnetic Kit - bacteria (Epicentre, $1968 / 24 preps). This is both labor-intensive and expensive ($91 - $175 per sample).
2. Pull out the poly-A plant mRNA, then make cDNA from this RNA pool (bacterial mRNA, bacterial rRNA, and plant rRNA), use DSN to remove the abundant ribosomal fractions, and make libraries from what remains. This may not be very efficient.
I'll continue to search for other options, but so far the only other one I've found (a kit called mRNA-ONLY) is no longer available, so it probably wasn't very efficient.
Thank you very much for any help!
1. Bacterial mRNA can't be pulled out of a mixed pool, so we'd first have to pull out the poly-A plant mRNA (using poly-dT beads from Illumina kit), then pull out the plant rRNA (and plastid RNA) using either RiboMinus Plant kit (Life Technologies, $562 / 8 preps) or RiboZero rRNA Removal kit-Plant Seed/Root (Epicentre, $554 / 6 preps), then pull out the bacterial rRNA using either RiboMinus Transcriptome Isolation Kit - bacteria (Life Technologies, $493 / 12 preps), MICROBExpress bacterial mRNA enrichment kit (Life Technologies, $420 / 20 preps), or RiboZero Magnetic Kit - bacteria (Epicentre, $1968 / 24 preps). This is both labor-intensive and expensive ($91 - $175 per sample).
2. Pull out the poly-A plant mRNA, then make cDNA from this RNA pool (bacterial mRNA, bacterial rRNA, and plant rRNA), use DSN to remove the abundant ribosomal fractions, and make libraries from what remains. This may not be very efficient.
I'll continue to search for other options, but so far the only other one I've found (a kit called mRNA-ONLY) is no longer available, so it probably wasn't very efficient.
Thank you very much for any help!
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