Introduction to RNA-sequencing
Bulk RNA sequencing measures the average RNA expression profile of all cells in a sample. It can identify genes and pathways that differ between tissues, treatments, disease states, or other experimental groups. Because RNA from many cells is combined, the result represents a population average rather than the expression profile of each individual cell.
Bulk RNA-seq is well suited for differential gene-expression studies when individual cell identities are not required. It is often the most practical way to compare many biological replicates, purified cell populations, cultured cells, or relatively homogeneous tissues. It is also useful when the primary objective is pathway-level or gene-level expression rather than discovery of rare cell populations.
Bulk RNA-seq may obscure biologically important differences in a heterogeneous sample. An apparent expression change can reflect regulation within cells, a change in the proportions of different cell types, or both. Consider single-cell or single-nucleus RNA-seq when cell types, rare populations, cellular states, or cell-specific responses are central to the question. Standard poly(A)-selected mRNA-seq is also not designed to capture most microRNAs and other small RNAs.
Compatible sample types
Projects commonly begin with purified total RNA from cells, tissue, or other biological material. Very small sorted cell populations may be submitted directly into a validated low-input lysis workflow. Required quantity, concentration, volume, purity, and RNA integrity depend on the selected method. Contact the CoLab before preparing very low-input, degraded, fixed, or otherwise unusual material.
Genomic CoLab offerings
Universal Plus mRNA-seq: Stranded, poly(A)-selected library preparation for standard bulk gene-expression studies from adequate quantities of reasonably intact total RNA.
Universal Plus total RNA-seq: Stranded, library prep without poly(A)-enrichment but typically with either ribosomal depletion or hemoglobin depletion (or both) library preparation for bulk gene-expression studies from adequate quantities of reasonably
intact total RNA.
Low-input SMART-Seq/Nextera: An unstranded, poly(A)-selected library preparation for low-input bulk workflow for very small sorted cell populations (1-500 cells) or RNA quantities below the standard bulk workflow. Although it may begin with only a small number of cells, it produces a population-level measurement and is not a droplet single-cell assay.
See our bulk RNA-sequencing pricing.
Additional specialized RNA workflows are possible after consultation when the choices above are not suitable due to RNA degridation or tissue fixations. Please contact the Genomics CoLab for more information.
Design considerations
When you start your project you will discuss with Genomics CoLab term the following items to assure we can meet your project needs:
- Whether the project needs a population-average or cell-resolved result.
- Whether poly(A) selection is appropriate for the transcripts and sample quality.
- Number of biological replicates and avoidance of confounding between experimental groups and processing batches.
- RNA extraction method, quantity, purity, and integrity.
- Read length and sequencing depth needed for gene-level expression, transcript-level
questions, or other specialized objectives.
What the CoLab performs
Depending on the project, the CoLab performs sample quality review, library preparation, library quality control, normalization and pooling, sequencing coordination, and agreed-upon data processing or analysis. In certain cases the Genomics CoLab can preform RNA extractions from standardized tissue/cellular sources utilizing our Kingfisher Flex and Zymo Quick-RNA Magbead extraction kits. Please inquire for more details about what may be possible.
What the investigator supplies
The investigator supplies the biological samples or RNA, sample metadata, the experimental design and comparison groups, relevant reference-genome information, and any known sample limitations. Investigators are responsible for collecting and balancing biological replicates appropriately and for following the project-specific submission instructions supplied by the CoLab.
Data and analysis delivered
Deliverables include raw sequencing data, but may be expanded to include aligned reads, gene-level count tables, quality-control summaries, normalized expression results, differential-expression results, standard figures and pathway enrichment analysis. Exact deliverables and the scope of interpretation are agreed upon before the project begins.
Pricing and sequencing considerations
Costs depend on the library-preparation method, number of samples, processing model, sequencing depth, read configuration, and analysis scope. CoLab service fees, sequencing charges, upstream sample preparation, and optional analysis should be shown separately in an estimate. Larger batches can make more efficient use of library preparation and sequencing capacity, but experimental groups should remain balanced across batches.
To start a project
Please download our project description form, fill it in with the as much detail as you can and then send it along with a consultation request to the [email protected] email address.