What are the key considerations for pooling during sequencing?

Answer:
3' scRNA-seq libraries are low-diversity libraries with high AT content and require special consideration during pooling.

Pooling strategies:

• Pooling only 3' scRNA-seq libraries in one lane:
  Although base composition is unbalanced, the biased regions are typically not used for downstream analysis.
  In such cases, no PhiX or minimal PhiX (~1%) is sufficient. 
• Pooling with other low-diversity libraries (e.g., scMLPSeq libraries, SeekSpace spatial barcode libraries):
  These libraries can be pooled at arbitrary ratios without introducing new interaction effects, as all share similar bias characteristics. 
• Pooling with balanced libraries:
  Balanced libraries can compensate for base bias.
  In this case, low-diversity libraries should not exceed ~30% of total molar ratio to maintain sequencing quality.  

General recommendations:

• Avoid duplicate index sequences within the same lane 
• Convert concentration to molarity: 

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Prefer pooling libraries with similar fragment size distributions
(3' libraries typically peak at 350–750 bp)