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[[!pmidi 28623350 desc="1 µg total RNA reverse-transcribed with SuperScript III and 2 µM random primer (GTATAAGAGACAGNNNNNNN). The RNA strand degraded with 2U RNAse H for 20 mi at 37 °C. The DNA strand was purified with Agencourt AMPure XP beads. 0.6 mM of SL primer (TCAGTTTCTGTA) was annealed to 25 µL of DNA from the previous step in 1x NEB buffer at 98 °C for 5 minutes and cooled down to room temperature for min. Second-strand synthesis with 5U Klenow fragment and 0.4 mM dNTPs in 50 µL at 37 °C for 60 minutes."]]

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Reduction of rRNA or hemoglobin with as few as 40 primers, while keepign whole-transcriptome coverage, at the cost of as systematic bias.

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Single-cell RNA-seq transcriptome analysis of linear and circular RNAs in mouse preimplantation embryos.

'Single-cell universal poly(A)-independent RNA sequencing (SUPeR-seq).' Linkers added by poly-a tailing of first-strand cDNAs. Random primers but very low levels of rRNA reads.

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Differential priming of RNA templates during cDNA synthesis markedly affects both accuracy and reproducibility of quantitative competitive reverse-transcriptase PCR.

cDNA yield did not increase with higher concentrations of specific hexamers. Higher yields with random hexamers. No data for reverse-transcription without primers.

Hansen KD, Brenner SE, Dudoit S.

Nucleic Acids Res. 2010 Jul;38(12):e131. doi:10.1093/nar/gkq224

Biases in Illumina transcriptome sequencing caused by random hexamer priming.

The sequence bias is not observed in libraries fragmented after random priming.

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Consistent errors in first strand cDNA due to random hexamer mispriming.

Error profile on ERCC spikes.

Biotechniques. 2006 May;40(5):649-57.

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Reverse transcription using random pentadecamer primers increases yield and quality of resulting cDNA.

Almost 100% of the RNA molecules are primed (using massive amounts of oligonucleotides).

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Nat Methods. 2009 Sep;6(9):647-9. doi:10.1038/nmeth.1360

Digital transcriptome profiling using selective hexamer priming for cDNA synthesis.

Because of reduced complexity, many 5′ ends are difficult to cover.

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Increased specificity of reverse transcription priming by trehalose and oligo-blockers allows high-efficiency window separation of mRNA display.

Reverse transcriptase can prime when the last two bases mismatch.