What is the Best Practice for 5' scRNA-seq?

Prepare: General reagent handling for best practice

• Thaw buffers and master mixes on ice. Only equilibrate reagents at room temperature when specifically instructed (e.g., Barcoded Beads, Cleanup beads, size-selection beads). Avoid repeated freeze-thaw cycles.
• Enzymes (2× Master Mix, RT enzyme, ligase, fragmentation enzyme) are highly temperature-sensitive: frozen until use, keep on ice at all times during the experiment, and return to −20℃ immediately. 
• Never vortex enzymes or master mixes. Mix gently by pipetting.
• Small-volume pipetting: Insert the tip into the middle of the liquid, pre-rinse by aspirating/dispensing 1–3 times, then aspirate slowly. For viscous reagents, do not pre-rinse; instead, pipette slowly and hold the tip in the liquid for ~5 sec before withdrawal.
• For viscous reagents (RT enzyme, barcoded beads), pipette slowly and keep the tip just below the liquid surface to prevent bubbles or volume loss.
• Prepare fresh 80% ethanol on the day of use.
• When preparing master mixes for multiple samples, always calculate ~10% extra volume to compensate for pipetting loss.
• Use only low-retention, nuclease-free, DNase/RNase-free tubes and tips to prevent adsorption or contamination.

Step 1: Sample preparation

Best practice for Sample Handling

• Keep single-cell/nucleus suspensions on ice and process quickly:
  • Single-cell suspensions: prolonged exposure (>20 min) reduces cell viability.
  • Single-nucleus suspensions: prolonged exposure increases the risk of RNA degradation.
• For nuclei prep, always add an RNase inhibitor at a working concentration of 0.2 U/µL. Use ≤0.1% BSA in the resuspension buffer. Perform two-step QC: count with a fluorescence cell counter, confirm morphology by Trypan Blue staining under the microscope.
• In single-cell experiments, if cell viability is >85%, use the concentration of live cells (not total cells) for all calculations.

Best Practices for Cell Suspension Quality Control

To ensure high-quality single-cell suspensions suitable for downstream applications, the following criteria are suggested:

Step 2: Droplet Generation using SeekOne DD

Best practice of running SeekOne DD instrument

• Calibrate the pipette according to the instructions of SOP. Pipette accuracy is critical, especially when handling size selection beads.
• Pre-program thermal cyclers. If starting at 4 °C, pre-run to reach temperature before loading samples.
• Run SeekOne DD self-check before use. Ensure the instrument is level, vibration-free, and free of obstructions.

Best practice of Chip preparation:

• Assemble Chip S3, Chip Holder, and gasket before preparing the single-cell mixture.
• Install Chip P in all unused positions to ensure all 8 slots are filled.
• Align gasket correctly: the notched corner goes to the top-left, holes aligned with wells, and the surface with “SeekGene” text facing up. Do not touch the smooth surface.
• Keep the Chip Holder lid closed with gasket attached until loading samples to prevent contamination.
• Use Chip S3 within 24 h of opening to avoid contamination or performance loss.

Best practice of Mixing preparation:

• Add components in the order: (1) Master Mix; (2) nuclease-free water or resuspension buffer; (3) cells/nuclei. This minimizes stress on cells and ensures viability.
• Avoid adding nuclease-free water directly to suspensions. Always add water or an appropriate resuspension buffer before cells/nuclei.

Best practice of Barcoded beads handling:

• After thawing, vortex thoroughly, centrifuge briefly, and pipette slowly to avoid bubbles. Aspirate slowly and allow ~5 sec before removing the tip to ensure full transfer.
• Barcoded beads: Use one tube per sample. Thaw at room temperature for 30 min before use. Avoid >3 freeze–thaw cycles. If not used within 3 hours, return directly to −80℃ (not −20℃).

Best practice of Droplet handling: 

• Aspirate gently along the tube wall, pipetting slowly (~20 sec per transfer) to minimize bubbles.
• At the droplet recovery well (well #4), there is often excess carrier oil at the bottom. Carefully remove this oil before transferring the water-in-oil droplets, without disturbing the product. This helps control the final recovered droplets volume to ~120 µL, ensuring uniform heating during downstream PCR.
• Use a deep-well PCR block that can evenly heat ≥100 µL reaction volume. If only standard blocks are available, split the sample into multiple tubes for PCR, then pool them afterward. Be aware that splitting and pooling may increase sample loss.

Best practice of Handling Multiple Chips Simultaneously:

1. Prepare chip setup

• When processing multiple samples or performing the operation for the first time, first remove the required number of Chip S3 according to your sample count.
• Replace the corresponding positions of Chip P in the chip holder with the Chip S3 chips.
• Close the chip holder cover and mount the Gasket on the upper layer of the holder, ensuring that the gasket holes are aligned with the chip wells.
• This prevents dust contamination before sample loading.
• After setup, proceed to prepare the single-cell mixture.

2. Load reagents sequentially

• When handling multiple samples, add reagents to each chip position sequentially by reagent type:

1. Add single-cell mixture to Well 1 of each chip in sequence.
2. Add Barcode Beads to Well 2 of each chip in sequence.
3. Add Carrier Oil to Well 3 of each chip in sequence.

This sequential loading approach helps maintain consistency across chips and minimizes timing differences between samples.

Step 3  cDNA Amplification

Best practice of cDNA Amplification:

1. Before Starting: 

Spin droplets briefly before adding the Demulsion Agent to minimize sample loss.

2. Phase Separation Awareness: 

• After adding the Demulsion Agent, retain the pink aqueous phase (this is the target cDNA).
• Discard the clear oil phase carefully, avoiding aspiration of the upper aqueous layer.

3. Magnetic Bead Handling:

• Equilibrate both Cleanup Beads and Size Selection Beads to room temperature for 30 minutes before use. Using cold beads may cause turbidity in the aqueous phase, so ensure proper equilibration before mixing.
• Vortex beads for at least 2 minutes before use; during adsorption, gently pipette-mix 10–15 times.
• During centrifugation, orient the bead side of the tube outward (away from the rotor center) for efficient pelleting.
• Follow ethanol wash volumes precisely — these vary by step.
• Allow beads to air-dry to a dull/matte finish; avoid over-drying, which causes clumping and poor elution.
• After elution, ensure no beads remain in the supernatant before proceeding.

Step 4: Library Construction & QC

Best practice of library construction:

• Bead Handling: Gently pipette up and down 5× during bead adsorption to improve cDNA yield.
• Index PCR: Prevent index duplication when pooling libraries. Assign unique index combinations to each sample within the same sequencing batch.
• Quality Control (QC) Checkpoints: Repeat bead purification as needed, but note that additional purification may reduce yield, especially if library concentration is <3 ng/µL. Use 0.6×bead purification when concentration >30 ng/µL. Use 0.75× in most cases.
• Documentation: Proper labeling ensures accurate sample tracking throughout pooling and sequencing. Immediately after QC, clearly label each tube with:
• Sample name
• Index number
• Library concentration