Bayega A, Oikonomopoulos S, Wang YC, Ragoussis J.

Front Genet. 2022 Oct 17;13:1031355. doi:10.3389/fgene.2022.1031355.

Improved Nanopore full-length cDNA sequencing by PCR-suppression.

“We observed [a range of 2.2 to 8.3-fold increase in yield] of amplicons from the Panhandle method compared to the ONT method.” “Further, the cDNA profile of samples from Panhandle protocol showed a significantly reduced amount of molecules below 600 bp compared to ONT protocol.” “Reads generated with the ONT protocol showed a marked 3′ bias with only about 40–50% of reads showing full-length coverage of the genes”

for each sample total RNA was added together with

RNA + 1 μL of 10 μm oligo (dT) primer + 1 μL of 10 mm dNTPs + Final volume: 11.6 μL pre-RT reaction.

the reaction was incubated at 72°C 3 min. 4°C 10 min, 25°C 1 min, then held at 4°C.

A 10.4μL reverse transcription (RT) reaction containing 1 X Maxima H Buffer, 1 μL RNaseOut (NEB), 2 μL of 100 μm TSO, 2 μL of 5M Betaine (Sigma-Aldrich), and 1 μL of Maxima H reverse transcriptase was added to the pre-RT reaction and the reaction incubated as shown in Supplementary Protocol.

Following reverse transcription,

5 μL of cDNA was used in a 50 μL PCR reaction containing 1 μL of 10 μm PCR primer and 25 μL of 2x LongAmp Taq Master mix (NEB).

PCR was performed as shown in Supplementary Protocol. 20 PCR cycles were used. Following PCR, 1 μL of exonuclease (NEB) was added to each reaction and incubated for 15 min at 37°C followed by 15 min at 80°C and the samples were purified using 1x AMPure XP beads (Beckman Coulter).