Day 1 (steps 1-13)

Preparation of DNA-free RNA. TIMING 45 min

Phosphatase treatment. TIMING 4 h

Day2 (steps 14-41)

5′ Cap removal. TIMING 4h

Reverse transcription. TIMING 2.5 h

Generate second strand by PCR. TIMING 2.5 h

Day 3-4 (steps 42-52)

Optional size selection. TIMING 4 h

Produce sticky ends. TIMING 1.5 h

Circularize full-length cDNA. TIMING 19h

Day 4-5 (steps 53-94)

Sonicate circularized DNA. TIMING 1.5 h

Bind samples to streptavidin beads. TIMING 1 h

End repair DNA fragments. TIMING 45 min

Add a protruding A to the DNA fragments. TIMING 45 min

Ligation of barcoded Illumina adaptors. TIMING 1.5 h

Day 5-6 (steps 95-109)

Library PCR amplification. TIMING 1.5 h

Library size selection. TIMING 1.5 h