DNBelab C Series Single-cell 5'RNA & V(D)J Library Prep FAQs
DNBelab C Series Single-cell 5'RNA & V(D)J Library Prep - Frequently Asked Questions
What is the DNBelab C series High-throughput Single-cell 5'RNA&V(D)J Library Preparation Kit, and what do I need to provide myself?
Purchasing information is available on the official MGI Tech website, or by contacting the sales team directly. For instruments and reagents customers must supply themselves, refer to section 1.6 “List of Self-provided Materials” in the user manual. The droplet generator applies to altitudes up to 2,500m.
What is the difference between 5'-end and 3'-end capture, and why is 5'-end used for VDJ?
In 3′-end library construction, the terminal sequence of Cell Beads is poly(dT), which pairs with the polyA structure at the 3′ end of the transcript. In 5′-end library construction, the terminal sequence of Cell Beads is a switch oligo sequence with a polyG structure at the end; the transcript adds a polyC structure to its 5′ end under enzyme action to complete the pairing. Since the VDJ region sits at the 5′ end of the transcript, only the 5′ library construction method retains cell barcode information and enables single-cell level VDJ sequencing.
What sample types are applicable?
5′ RNA library preparation suits animal cells and cell nuclei. V(D)J library preparation has built-in enrichment primers for human and mouse TCR and BCR only; primers for other species must be self-designed. Typical samples include PBMC, spleen, and bone marrow cells.
What are the input requirements for cell or cell nucleus samples?
Diameter of cell/cell nucleus < 60 μm, viability > 80%, impurity and aggregation rate < 5%, and input cell number < 30,000 preferably (above that, a multiplet rate below 8% cannot be guaranteed). Samples outside these requirements may not achieve guaranteed data quality, and the risk is borne by the customer.
How should dead cells, cell clusters, and fragmented impurities be handled?
- Dead cells: Use the Dead Cell Removal Kit from Miltenyi Biotec, selecting the model based on cell input amount and dead cell ratio.
- Cell clusters/tissue blocks: Remove by enzymatic dissociation for an appropriate period, or by selecting a cell strainer with an appropriate aperture.
- Fragmented impurities: Depending on fragment type and proportion, use density gradient centrifugation or a kit-based method (e.g., Miltenyi Biotec’s Fragment Removal Kit or equivalent).
What is the cell capture efficiency?
Capture efficiency shall be ≥50% if the prepared sample meets specifications.
What is the injection sequence for droplet generation, and what is the multi-bead capture principle?
The injection sequence Cells → P100 Oil → Beads must be followed; the cell phase must enter the inlet first to prevent the oil or bead phase from entering the cell channel prematurely, which would compromise droplet quality and stability. MGI’s multi-bead capture technology uses two functional bead types per effective droplet: one or more Cell Beads (large beads) and multiple Index Carriers (small beads). Cell Beads carry long sequences with a GGG tail structure to capture cDNA after reverse transcription, plus short sequences for pairing with the Index Carriers. Index Carriers merge mRNA information released by the same cell across multiple Cell Beads, improving recognition accuracy and capture efficiency versus platforms affected by barcode multiplets (Lareau et al., Nat Commun, 2020, 11:866).
How can I confirm droplet generation went normally, and is cross-contamination a risk between runs?
Under normal conditions there’s no liquid leakage from the chip-on-board, so no cross-contamination occurs between successive samples even if the same position is reloaded. If liquid spills onto the carrier due to misoperation, clean with 75% alcohol; collection hole sealing pads are single-use. To confirm normal generation, observe residual liquid in the sample-loading well after completion (cell phase should be empty or near-empty), then sample droplets from the collection hole, dilute in P100 Oil on a slide, and check under an inverted microscope for uniformly sized droplets.
At what step are the Index Carrier sequences released, and are there stopping points during the workflow?
After droplet formation, the DIR Reagent in the cell phase releases the sequence on the Index Carrier into the droplet, where it’s captured by complementary pairing primers on the Cell Beads. On the first day, testing can be stopped once reverse transcription begins, with samples collected the next day to continue. Refer to the user manual for other stopping points.
How is barcoding handled across cDNA, TCR, BCR, and Oligo libraries, and what PCR settings are required?
If four libraries are sequenced together on one MGISEQ-2000 lane or DNBSEQ-T7 chip-on-board, different barcodes must be used for each so the sequencer can separate them by sample tag. If libraries aren’t sequenced in the same channel, barcodes can be freely chosen (1-16 for 4-reaction kits, 1-32 for 16-reaction kits); for more than 32 barcodes, purchase the “DNBelab C-series Single-cell Library Preparation Sample Tag Kit H” (item No. 940-001978-00) separately. During adapter ligation for cDNA, TCR, and BCR library construction, the PCR instrument’s hot cover mode must be turned off due to the ligase’s low working temperature (the cover can be opened if it’s above 25°C).
Can the immune repertoire library be built without the transcriptome library, and can libraries be pooled during cyclization?
No — immune library data analysis requires the 5′ RNA transcriptome data to merge large magnetic beads, so a transcriptome library must always be constructed alongside it. During single-strand cyclization, up to 4 libraries of the same type (cDNA, TCR, BCR, or Oligo) can be pooled and circularized together, but different library types must not be pooled and circularized together, as this would affect subsequent DNB preparation.
Why is nested PCR used for TCR/BCR enrichment, and can the full-length variable region be obtained?
Nested PCR is used because if the first amplification produces an erroneous fragment, the probability of that fragment being paired and amplified again in the second round is extremely low — reducing false positives. Specific amplification primers designed against the TCR and BCR constant regions enrich and sequence the entire V(D) gene fragment, enabling full-length variable region recovery.
Is it necessary to change sequencing primers, and can libraries be pooled for sequencing?
No primer changes are needed — the kit is only compatible with MGI sequencers (MGISEQ-2000, DNBSEQ-T7, DNBSEQ-T20) and sequencing reagents (PE100, PE150), using the same adapters as other MGI library types. A balanced library is not required. A dark reaction must be set up (refer to the DNBSEQ-T7/MGISEQ-2000 ECR V7.0 manual or contact MGI support for sequencing scripts). cDNA, TCR, BCR, and Oligo libraries can be pooling-sequenced together; recommended DNB mass pooling ratios and resulting actual output ratios vary by case (e.g., cDNA:Oligo≈12:1 alone, or cDNA:TCR&BCR:Oligo≈4:8:1 combined) — contact MGI technical support for guidance specific to your sample count and library mix.
