We are trying to FACS sort shh-responsive cells from injured mouse facial nerve and run RNA-seq on them. I have limited bench experience. Any advice or detailed protocols for these procedures? I appreciate the help.
If you have very low cell counts after sorting and obtained limited RNA quantity, NEB just demoed us with their new NEBNext Ultra II Directional RNA library Prep Kit for Illumina (Ribosomal Depletion method and works with very low input >20ng). Price also pretty good since they make their own enzyme. You get the kit and follow their protocol. We got pretty good quality library both coverage and quality.
Our lab has always used Illumina Truseq Stranded mRNA prep kit which is always very reliable. Illumina is a little more expensive than others, requires good amount of input material.
Main thing when try to prepare RNA library is quality check, i.e. to make sure your input RNA has good integrity (high RIN number, kit usually say >8, but my PI has always wanted ~10). You do not want "garbage in, garbage out".
These Kits won't come with reversetranscriptase or beads, so you need to get the Ampure beads and Super Script III or II.
Hello Alireza,
I infer that you wish to isolate motor neurons from injured versus non-injured animals. In my opinion, the most important aspect is to standardize the dissociation procedure. This largely depends on the species and age of the animals: neonates, adults?
In my lab we have developed several protocols for FACS-isolation of motor neurons from both rodents and humans (iPSc) suitable for various types of analysis. See for instance this published ms available on ResearchGate. Other papers will come out soon. We have also protocols working for Schwann cells of peripheral nerve.
https://www.researchgate.net/publication/279070006_Modeling_Amyotrophic_Lateral_Sclerosis_in_Pure_Human_iPSc-derived_Motor_Neurons_Isolated_by_a_Novel_FACS_Double_Selection_Techniqu
Please let me know whether this post was helpful or whether you like me to provide further advice.
Good luck.
Best wishes
Georg
Article Modeling Amyotrophic Lateral Sclerosis in Pure Human iPSc-de...
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