The recommended input range is 10 - 500 ng of total RNA. No prior poly(A) enrichment or rRNA depletion is required.
The amount of total RNA needed for QuantSeq depends on the poly(A) RNA content of the sample in question. The protocol was tested extensively with various cell cultures, animal and plant tissues, yeast, fungi, drosophila, and human reference RNA.
As a starting point, we recommend performing the protocol initially with 500 ng total RNA. RNA inputs ≥200 ng are recommended to detect low abundant transcripts efficiently.]
If you are limited in terms of input, we recommend using QuantSeq FWD. By choosing longer sequencing read lengths, you are also able to read into the 3' ends.