Genome scaffold ORF A   ORF B RNASeq reads Without a strand specific library, you can’t know from which ORF the reads from the overlapping region arose.

mRNA

mRNA Fragmented mRNA

generate 1st cDNA strand Add random hexamers to generate 1st cDNA strand ATGGTC

Add RNAs to digest RNA

More random hexamers to generate 2nd cDNA strand. Include dUTP instead of dTTP. AAGGAC

More random hexamers to generate 2nd cDNA strand. Include dUTP instead of dTTP. U U U

Ligate Y-shaped linkers/adapters U U U

Digest UTP-containing strand

First round of PCR

First round of PCR

Second round of PCR

Second round of PCR

Sequencing library

Second round of PCR The sequences are different at the left and right ends. That means, if you sequenced from the right, you’re getting mRNA reads from ORF B. If you sequenced from the left, you’re getting reads from ORF A. Genome scaffold ORF A   ORF B RNASeq reads