What are the nucleotide sequences of the adapters required for trimming prior to the software analysis of the sequencing reads?

The final cDNA library generated with the SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian carries Illumina TruSeq® HT (currently known as TruSeq CD) indexing primers. The nucleotide sequences of the TruSeq HT i5/i7 indexes are available in the SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian User Manual. The complete nucleotide sequences of the corresponding Illumina [...]

Will collected cells change the volume of the cell collection buffer?

Potentially. The collected cell volume will depend on the cell diameter, so we recommend performing a preliminary cell sorting pilot study: sort 50, 100, and 1,000 cells using the appropriate nozzle and pressure settings to determine the transfer volume of the sheath fluid. If this varies from the predicted transfer volume, you may wish to adjust the volume of cell collection buffer.General [...]

How should cells be collected for cDNA synthesis?

For cDNA synthesis directly from cells, it is essential that the cells remain undamaged during cell collection, as damaged cells may contain compromised DNA that can act as a template for reverse transcriptase and contaminate the final cDNA library. If feasible, we recommend verification of cell integrity prior to FACS collection. Please note that the low-pressure setting should be used on the [...]

What are abnormal Bioanalyzer traces for SMARTer stranded kits?

All stranded kits: Few reads from the sequencing run, or few clusters passing filter. SMARTer stranded libraries can have a lower than average pass-filter rate due to low complexity for the first three cycles. Illumina software has problems interpreting low complexity libraries. Decreasing the cDNA library loading concentration and/or spiking in 5–10% PhiX control DNA (Illumina) may correct this [...]

What are abnormal Bioanalyzer traces for SMARTer Ultra low kits?

The electropherogram shows a broad size distribution often with multiple small peaks. This is characteristic of a degraded RNA input sample. You may need to gather new RNA samples if you proceed with SMARTer Ultra low kits.An electropherogram of cDNA generated from degraded RNA template. The broad size distribution and multiple small peaks indicate poor-quality cDNA generated using the SMARTer [...]