Sample Preparation

The Illumina Single Cell 3′ RNA Prep protocol requires a suspension of high-quality single cells or nuclei as input derived from cell cultures, dissociated tissues, cell sorting, or other isolation methods.

The following guidelines apply to sample preparation:

• Minimize the presence of dead cells or aggregates to ensure the highest quality data.
• Minimize the amount of time that it takes for sample preparation through capture and lysis, and add RNase inhibitor at 0.4–1 U/µl final concentration to buffers being used in lengthy steps (dissociation, enrichment, sorting collection tubes, or sample suspension buffers).
• Use higher RNase inhibitor concentrations for challenging sample types.
• When processing many samples together at once, Illumina recommends processing sample preparations in batches through capture and lysis to prevent mRNA degradation.

Depending on your input type, refer to the Prepare Cell Suspension or Prepare Nuclei Suspension for sample preparation instructions. Start capture and lysis as soon as possible after diluting samples to the target concentration.

Fixed cells generated according to DSP-Methanol Fixation for Cells Protocol Instructions are compatible with this workflow. If cells are in media containing FBS or BSA, complete a 1 ml wash step using Cell Suspension Buffer, then resuspend in Cell Suspension Buffer that does not include additional BSA or RNase inhibitor before starting the fixation protocol. This fixative yields comparable performance as fresh cells. Fixed cells can be stored for up to seven days at -25°C to -15°C. Alternative fixation approaches (eg, formaldehyde) have not yet been validated and are not recommended.

Fixed nuclei are compatible with this workflow. Before starting the fixation protocol nuclei must be resuspended in Nuclei Suspension Buffer (NSB) without BSA or RNase inhibitor. Refer to DSP-Methanol Fixation for Nuclei Protocol Instructions for instructions on fixing isolated nuclei. You are strongly advised to substitute NSB working solution for the wash steps in the alternative nuclei fixation protocol (at least 1 ml of NSB working solution without BSA or RNase inhibitor in up to two wash steps and the final resuspension before fixation). Fixed nuclei can be stored for up to two months at -25°C to -15°C.