Choanoflagellates are co-cultured with their bacterial food source. PolyA selection can be used to separate choanoflagellate mRNA from bacterial mRNA, because bacterial mRNA is not polyadenylated. Using total RNA prepared from a culture of Salpingoeca rosetta grown with the prey bacterium Algoriphagus machipongoensis, we tested whether repeating the polyA selection steps in the standard Illumina TruSeq v2 mRNA preparation protocol would lead to increased separation of choanoflagellate from bacterial mRNA. The repeated (four-round) method removed roughly an order of magnitude more non-polyadenylated RNA than the standard (two-round) method, with no significant loss in S. rosetta transcript coverage.