RNA-seq from 500 L1s

Model Organism: Worms Methods & Reagents
1

Hi all,

Is it possible to do RNA-seq from 500 L1s? What RNA isolation method, processing (Poly-A selection or rRNA depletion), library building kit would you use?
I am really restricted to the amount of worms I have, the max I can get is 1000 L1s.

Thank you!!

2

hello,

might i ask why you are limited to 1000 L1s? are you working with a balanced lethal gene?

have you thought about using large scale RNAi? ive done it on up to 100 plates before, and i can harvest roughly 1ml of packed worms.

3

Yes, that’s more than sufficient. Several prep kits (e.g., NEB’s Ultra II, w/ polyA purification) require only 5-10 ng total RNA as input.

4

You can totally get a great prep from those numbers.

With FACS sorted pooled cells I can get great RINs with just a few thousand cells. I use TaKaRa’s SmartSeq v4 kit combined with the Illumina Nextera XT kit for library prep. Works phenomenally.