Step Overview (Step 8.1d)

Correlation between input & library complexity

A Single Cell Gene Expression library is generated using a fixed proportion (10 μl, 25%) of the total cDNA (40 μl) obtained at step 7.2n. The complexity of this library will be comparable to one generated using a higher proportion (>25%) of the cDNA. The remaining proportion (30 μl, 75%) of the cDNA may be stored at 4°C for up to 72 h or at −20°C for longer-term storage (up to 4 weeks).

Note that irrespective of the total cDNA yield (ng), which may vary based on cell type, targeted nuclei recovery etc., this protocol has been optimized for a broad range of input mass (ng), as shown in the example below. The total number of SI PCR cycles (step 8.5d) should be optimized based on carrying forward a fixed proportion (10 μl, 25%) of the total cDNA yield calculated during Post cDNA Amplification QC & Quantification (step 7.3).

Example: Library Construction Input Mass & SI PCR Cycles

Cell Type	Targeted Nuclei Recovery	Total cDNA Yield (ng)	cDNA Input into Fragmentation		SI PCR Cycle Number
			Volume (µl)	Mass (ng)
High RNA Content		150 ng	10 µl	37.5 ng	14
		400 ng	10 µl	100 ng	13
Low RNA Content		1 ng	10 µl	0.25 ng	16
		100 ng	10 µl	25 ng	14

The term "cell" as used here applies to nuclei too.