In Mortazavi et al's paper entitled with "Mapping and quantifying mammalian transcriptomes by RNA-Seq", it was described that:
I am just wondering the cDNA preparation method is followed by the protocol provided by Illumina? Thanks in advance!
The mRNA was first fragmented by addition of 5× fragmentation buffer (200
mM Tris acetate, pH 8.2, 500 mM potassium acetate and 150 mM
magnesium acetate) and heating at 94 °C for 2 min 30 s in a thermocycler
and was then transferred to ice and run over a Sephadex-
G50 column (USA Scientific) to remove the fragmentation ions.
The reason for using random priming rather than dT priming is
that the latter typically produces a bias in the product that favors
3′ end representation. The extension issues do not affect all transcripts
identically, and the cDNA population that results for each RNA species
is a complex function of its specific properties as a substrate for
reverse transcription and the overall transcript length. We used 3 μg
random hexamers, added to prime first-strand reverse transcription
according to the manufacturer’s protocol (Invitrogen cDNA synthesis
kit). After the first strand was synthesized, a custom secondstrand
synthesis buffer (Illumina) was added, and dNTPs, RNase H
and Escherichia coli polymerase I were added to nick translate the
second-strand synthesis for 2.5 h at 16 °C. The reaction was then
cleaned up on a QiaQuick PCR column (Qiagen) and eluted in 30 μl
EB buffer (Qiagen).
mM Tris acetate, pH 8.2, 500 mM potassium acetate and 150 mM
magnesium acetate) and heating at 94 °C for 2 min 30 s in a thermocycler
and was then transferred to ice and run over a Sephadex-
G50 column (USA Scientific) to remove the fragmentation ions.
The reason for using random priming rather than dT priming is
that the latter typically produces a bias in the product that favors
3′ end representation. The extension issues do not affect all transcripts
identically, and the cDNA population that results for each RNA species
is a complex function of its specific properties as a substrate for
reverse transcription and the overall transcript length. We used 3 μg
random hexamers, added to prime first-strand reverse transcription
according to the manufacturer’s protocol (Invitrogen cDNA synthesis
kit). After the first strand was synthesized, a custom secondstrand
synthesis buffer (Illumina) was added, and dNTPs, RNase H
and Escherichia coli polymerase I were added to nick translate the
second-strand synthesis for 2.5 h at 16 °C. The reaction was then
cleaned up on a QiaQuick PCR column (Qiagen) and eluted in 30 μl
EB buffer (Qiagen).
