Single-cell RNA-seq Analysis

This README describes the output we provide upon running Cellranger and other preliminary analyses for your single-cell RNA-seq data. It also suggests next steps of the analysis that can be done by you and by us.

Documentation of the analysis output

There is a zip file for each sample, and in case of multiple samples, a zip file containing the combined results (“all”).

YOU CAN FIND A COMPREHENSIVE DESCRIPTION OF THE CELLRANGER OUTPUT HERE.

Cellranger report

• Location: <sample>/web_summary.html
• To get started, take a look at the web_summary.html file, which includes an overview of the cell quality.

Loupe Cell Browser

• Location: <sample>/cloupe.cloupe
• Moreover, you can explore your data in the Loupe Cell Browser.
• You need to download and install the Loupe Cell Browser, and load the cloupe.cloupe file.
• The program is intuitive to use, and you can find a good tutorial here.

HTO demultiplexing (only if applicable)

• Location: <sample>/demultiplexed/
• If you have used hash tag oligos (HTOs) to pool multiple samples in one library, we have run our in-house demultplexing script to split the samples.
• In this case, we generated an *scrnaseq_hto.html HTML report, which provides an overview for your data, for example how many cells we identified for each sample.
• Moreover, the cell_classification_for_loupe.csv file can be imported into the Loupe Cell Browser to distinguish between the pooled samples.
• Last, counts per sample were written to file using the Cell Ranger format, so they can be used for follow-up analyses in R and python.

Velocyto

• Many of our clients expressed the wish to do trajectory analysis via RNA velocity in R and python. We therefore generated a loom file with exon and intron counts.
• Location: <sample>/velocyto/
• Not available for fixed RNA workflows

What we can do next

Comprehensive analysis

• QC and pre-processing, clustering, visualization, cell cycle effects, marker gene detection, differential expression analysis, functional enrichment analysis
• Output: Extensive report, Excel files for genes and enrichments, data export so you can further explore the data on your own
• Costs: 3 units

Additional advanced analyses

• Adding custom gene to reference
• Reference mapping
• Trajectory and velocity analysis
• Additional visualizations for publication
• Cell to cell communication
• Costs: individual cost estimates based on the required time