Differences between V4.0PLUS PIPseq and Illumina Single Cell (formerly PIPseq V)
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The high level advantages of Illumina Single Cell (Formerly PIPseq V) when compared to PIPseq V4.0PLUS are covered in this article while the full details are covered in the in the results section.
High-level benefits:
New sequencing-artifact reducing PIP design for more usable data.
New optimized chemistry includes novel reverse transcriptase that improves reverse transcription and template-switching efficiencies.
Increased sensitivity from using the full cDNA yield for library preparation after limited-cycle amplification of the cDNA (5 cycles only).
The random enzymatic fragmentation during library preparation results in Intrinsic Molecular Identifiers rather than UMIs to remove duplicates.
10 bp indexes that are pre-combined in a strip frame for multi-channel pipetting ease.
The Clean Up Library steps use a single-sided size selection (no double-sided size selection required).
The PIPseq platefuge microcentrifuge requirement for processing T2 reactions has been eliminated, but labs may continue to use it if preferred.
Protocol Changes:
Changes to master mix volumes for cDNA Synthesis and Amplify cDNA.
Assess cDNA Quality steps are completed after Clean Up cDNA using leftover supernatant. Leave the tubes on the magnetic rack and do not discard stock reagents used for Amplify cDNA, as they will be used again for the QC.
Library Preparation steps must be started within 72 hours of Clean up cDNA. Longer storage of limited-cycle cDNA can result in cDNA degradation.
There are changes to the Fragmentation, End Repair and A-Tailing, and the Ligation thermal cycler profiles; be sure to update these if the v4.0PLUS programs are saved.
The WTA primer used for Amplify cDNA must be diluted at a 1:10 ratio before adding it to the master mix for T2 kits only. It should not be diluted during Assess cDNA Quality.
For any feedback or questions regarding this article (Illumina Knowledge Article #9448), contact Illumina Technical Support .