Flow cell and cycle kit options for Illumina Single Cell 3’ RNA Prep libraries

Below is a table showing the estimated number of Illumina Single Cell 3’ RNA Prep samples that can be sequenced per flow cell, based on kit size and sequencing platform:

Estimatedᵃ Sample Throughput per Flow Cell

a. Calculations are based on the recommended sequencing depth per kit size. The recommended sequencing read depth for the T2, T10, and T20 kits is 20,000 reads per input cell (RPIC), while the T100 kit recommendation is 10,000 RPIC.

For additional guidance, see Why is Illumina Single Cell sequencing read depth based on input cells? and Cell input and volume loading recommendations for Illumina Single Cell 3’ RNA Prep

b. P3 and P4 flow cells only available on the NextSeq 2000 System.

c. Illumina Single Cell Unique Dual Indexes offers 96 indexes for 96 samples. Running over 96 samples per flow cell is possible with individual lane loading. Use the NovaSeq 6000 Xp workflow for individual lane loading on the NovaSeq 6000 System.

Recommended Sequencing Cycle Kits

100-Cycle Kit (Minimum Required Cycles):

Illumina Single Cell 3’ RNA Prep libraries can be run on a 100-cycle kit, as it provides 138 cycles of SBS. If using a 100-cycle kit use the following recipe: 45 + 10 + 10 + 72 = 137 cycles total.

150-Cycle Kit:

Illumina recommends maximizing the cycle number for Read 2 (cDNA read) when using a 150-cycle paired-end kit.

NOTE: Illumina Single Cell 3′ RNA Prep libraries are dual-indexed with 10-base i5 and i7 indexes. Read 1 (≥ 45 bases) contains the barcode information while Read 2 (≥ 72 bases) contains the gene expression information.

For details on read length, depth, final loading concentrations, and PhiX spike-in recommendations, refer to Illumina Single Cell 3' RNA Preparation library sequencing recommendations and guidelines and the Training Packet.

For any feedback or questions regarding this article (Illumina Knowledge Article #9567), contact Illumina Technical Support [email protected].