DNBSEQ-G400 sequencing; Deep sequencing of synthetic RNA (complex molecular spike-in set)
Deep sequencing of synthetic RNA (complex molecular spike-in set)
384 replicates of 10 ng synthetic spike-in RNA no further extraction was performed 384 replicates of 10 ng spike-in RNA each were reverse transcribed using the Smart-seq3 protocol at 1/10 volumes and pre-amplified for 8 cycles of PCR. Resulting cDNA was pooled and cleaned using 0.8x 22% PEG beads. Next, we amplified short 100-150 bp amplicons around the TSS using the TSO-specific Smart-seq3 FWD primer containing the Nextera Read 1 overhang and a sequence specific reverse primer for each of the 11 distinct spike-in sequences with a TruSeq Read 2 overhang.