We're trying to establish VASA-seq, an RNA sequencing protocol (Salmen et al. 2022, Nature) in our lab. Although having a sufficient amount of amplified RNA (around 100 ng/ul) after in vitro transcription (which is reduced to around 20 ng/ul after ribosomal RNA degredation), our PCR yield is too low on Qubit (around 1 ng/ul). I have tried using random hexamer instead of adapter ligation, RNase H without seeing any improvement. We have no clue why this is happening.

A short description of VASA-seq: RNAs is fragmented at 85°C in the presence of Mg2+. Then the 3'-ends are repaired and polyadenylated. Then CEL-seq2 primers are annealed to the poly-A tail. The following steps are as follows: reverse transcription--> 2nd strand synthesis -> in vitro transcription -> ribosomal RNA depletion -> adapter ligation (RA3) -> 2nd reverse transcription (with RTP) -> PCR (with RP1 and RPiX primers). I didn't mention a couple of small steps like ExoI treatment, RNase treatment, etc.