Questions & Answers
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General Single Cell RNA-seq
Common Single Cell Gene Expression and Immune Profiling Q&A
High Throughput Single Cell Gene Expression + Single Cell Immune Profiling
- What is the expected liquid volume in each well after a successful HT run?
- What dry baths are supported for use with HT?
- Are there differences in microfluidic failure rates (clogs and wetting failures) between the standard and HT chips?
- How do I assemble HT Gel Beads in the HT Gel Bead holder?
- What comes in my HT kit?
- Why do I have a 16 reaction library preparation kit with my 8 reaction HT kit?
Sample Prep - Cells
- What metrics should I be looking at to optimize my sample prep in my pilot experiment?
- Should I use warm or cold tissue dissociation?
- What are the best practices for flow sorting cells for 10x Genomics assays?
- Are there considerations for small cells or cells with low RNA?
- Are fresh frozen tissue samples compatible with Single Cell RNA sequencing?
- Can viral transcripts be detected with the Single Cell Assays?
Feature Barcoding - Cell Surface Protein
- What was updated in the Demonstrated Protocol for Cell Surface Protein labeling CG000149 RevC?
- How can I optimize my TotalSeq™ antibody labeling protocol?
- High fraction of reads coming from barcodes with very high UMI counts
- Are the feature barcode oligo conjugation's random nucleotides (Ns) required in the feature barcode oligo conjugation?
- How do I use the Feature Barcode whitelist?
- Can I do custom oligo conjugation for Feature Barcoding assays?
Feature Barcoding - Cell Multiplexing
- What are the differences between the tube-based and plate-based CMO labeling protocols for using the 3' CellPlex kit for Cell Multiplexing?
- Can antibody labeling be performed after CMO labeling when using the 3' CellPlex kit for Cell Multiplexing?
- Can I perform shallow sequencing on 3’ Cell Multiplexing libraries to assess the quality of my CellPlex data?
- How does high background from unbound CMOs affect Cell Multiplexing metrics in 3’ CellPlex experiments?
- How can I reduce background from unbound CMOs using the 3’ CellPlex kit for Cell Multiplexing?
- Which nuclei isolation protocols are supported for use with the 3' CellPlex Kit for Cell Multiplexing?
GEM Generation & Barcoding
- How should I take photographs to document clogs and wetting failures?
- How can I test whether my cells can be partitioned into GEMs?
- Is it possible to capture microRNAs in the Gene Expression assays?
- Can you run Single Cell 5' and 3' samples on the same Single Cell A Chip?
- What is a template switch oligo (TSO)?
- What is the maximum number of cells that can be profiled?
Post GEM-RT Cleanup & cDNA Amplification
- Can I store purified cDNA beyond 1 week at -20°C?
- Why is my total cDNA yield low?
- Why is the aqueous layer cloudy after breaking GEMs?
- What types of RNA can be detected?
- What are the additional peaks in my post-cDNA amplification QC?
- What is the expected size range for amplified cDNA?
- If full length mRNA is transcribed, how is the assay biased to 3' or 5'?
- Are 10x Single Cell gene expression libraries strand-specific?
- What are the additional peaks in my Single Cell Gene Expression library?
- Is there a safe stopping point between Post-Ligation Cleanup and SI-PCR?
- Is there a sequence preference during cDNA fragmentation?
- Can ERCC spike-ins be used for normalization?
- How do I pool 10x libraries for Illumina sequencing?
- Should I select Workflow A or Workflow B for the i5 index sequence?
- Can I sequence longer than 91-98bp on Read 2 for Single Cell 3' and 5' Gene Expression libraries?
- Can I decrease the sequencing length of Read 2 for Single Cell Gene Expression assays?
- Why do I see high levels of Malat1 in my gene expression data?
- How do I pool libraries with different target cell numbers?
- What do the percentages mean in the barcode rank plot?
- Is it required to sequence more for nuclei compared to cells ?
- Should I include introns for Targeted Gene Expression data analysis?
- Can I aggr data run with and without introns?
- Can I sequence less if I use intronic mode?
- I do not want to include intronic reads in my analysis. Can I do that in Cell Ranger?