What are general considerations for cDNA synthesis directly from cells?

Illumina indexes and adapters are integrated during cDNA amplification with the SMARTer Stranded RNA-Seq Kit (including the HT version) and the SMARTer Stranded Total RNA Sample Prep Kit - HI Mammalian. No additional library preparation is needed. (see ""Which indexes are included in SMARTer stranded kits?""). Not all indexes can be pooled together, consult the Illumina literature (such as the [...]

How do I shear rRNA-depleted RNA?

For cDNA synthesis using the SMARTer Universal Low Input RNA Kit for Sequencing (Cat. # 634938), we recommend shearing at the same time as priming the RNA. To do this, add 4 µl of 5X First-Strand Buffer to your RNA and the 3' SMART N6 CDS Primer II A (not to the Master Mix), then incubate at 94°C. For RNA with a RIN >7, incubate for 5 min. For RNA with RIN 4–7, incubate for 4 min. For RNA [...]

What ribosomal RNA depletion methods do you recommend?

For 10–100 ng samples of mammalian total RNA, we recommend using the RiboGone - Mammalian kit (Cat. # 634846) for rRNA depletion. General [...]

Why do I need to remove ribosomal RNA?

Since the SMARTer Universal Low Input Kit for Sequencing (Cat. # 634938) utilizes random priming for first-strand cDNA synthesis, if rRNA is not depleted, up to 90% of sequencing reads are expected to map to rRNA. General [...]

Which RNA purification kits are compatible with the SMARTer Universal kit?

There are many RNA extraction and purification methods compatible with the SMARTer Universal Low Input RNA Kit for Sequencing (Cat. # 634938). When choosing a purification method, ensure that it is appropriate for the particular sample type and the amount you are working with.Input RNA should be free of genomic or carrier DNA, and free of contaminants that would interfere with oligo-RNA template [...]