Bulk RNA sequencing is the method of choice for transcriptomic analysis of pooled cell populations, tissue sections, or biopsies. It measures the average expression level of individual genes across hundreds to millions of input cells and is useful to get a global idea of gene expression differences between samples.
At Single Cell Discoveries, we can offer normal and low-input bulk RNA sequencing with our sample multiplexing protocol. We use CEL-seq2-type barcodes to uniquely label each sample and subsequently pool (multiplex) samples together for library preparation.
Bulk RNA sequencing workflow
When bulk RNA sequencing samples are processed, we perform the following reactions.
- If the samples are TRIzol samples, our lab team will first extract the RNA.
- We perform pre-experimental quality controls*:
- The quality of the total RNA (of a number of selected samples) is measured with the Agilent Bioanalyzer system;
- The concentration is determined with Qubit;
- All samples are brought to the same concentration (normalization), to ensure that each sample receives an equal amount of reads during sequencing.
- A unique primer is added to each separate bulk sample.
- Reverse transcription and second-strand synthesis are performed to generate cDNA from the RNA.
- The bulk samples are now uniquely barcoded and will be pooled into one library. After sequencing, it is possible to distinguish all samples based on their unique barcode.
- The material of the pooled sample is amplified via an IVT reaction (linear amplification), which generates amplified RNA.
- The quality and concentration of the amplified RNA (aRNA) is determined by running the material on an Agilent Bioanalyzer system.
- Library preparation is performed to generate a sequenceable cDNA library.
- The quality and concentration of the cDNA library are determined by running the material on an Agilent Bioanalyzer system.
Read more about our bulk RNA sequencing service on this page.
*Important notice! If samples contain few cells, it is not possible to perform these quality controls, due to the low RNA yield. It is also not possible to normalize the samples. In these cases, our lab team will continue to the processing phase without the quality controls.