1. 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.

3. mRNA

4. mRNA
Fragmented mRNA

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

6. Add RNAs to digest RNA

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

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

9. Ligate Y-shaped
linkers/adapters U U U

10. Digest UTP-containing strand

11. First round of PCR

12. First round of PCR

13. Second round of PCR

14. Second round of PCR

15. Sequencing library

16. 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