Improvements to the nanoCAGE protocol. (A) Schematic shows how template switching concatemers form during the template switching reaction and how they interfere with sequencing. Concatemers are blocked by the addition of an 18-carbon spacer between the two 5′-most bases on the template switching oligo. (B) Both 5′ isomer (Iso) bases and an internal 18-carbon spacer (I18CS) were effective at reducing the percentage of reads that included concatemers. The addition of manganese to the template switching reaction increased the percentage of reads that matched concatemers. (C) The addition of Mn to the template switching reaction significantly increased the number of contaminating rRNA reads in the final library. Lines on graph represent means.