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What is Bulk RNA sequencing?

Bulk RNA-sequencing is the method of choice for transcriptomics 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 differences in gene expression between samples.
At Single Cell Discoveries, we can offer cost-effective bulk RNA sequencing due to our sample multiplexing protocol. We use CEL-seq2 type barcodes to uniquely label each sample and subsequently pool (multiplex) samples together for library preparation. This reduces the use of expensive reagents, which saves on costs.

Bulk RNA sequencing workflow

When bulk RNA sequencing samples are processed, we perform the following reactions:

  1. If the samples are TRIzol samples, our lab team will first extract the RNA.
  2. Pre-experimental quality controls are performed*:
    1. The quality of the total RNA (of a number of selected samples) is measured via the Agilent Bioanalyzer system
    2. The concentration is determined via Qubit
    3. All samples are brought to the same concentration (normalization), to ensure that each sample receive an equal amount of reads during sequencing
  3. A unique primer is added to each separate bulk sample.
  4. Reverse transcription and second-strand synthesis is performed to generate cDNA from the RNA.
  5. 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.
  6. The material of the pooled sample is amplified via an IVT reaction (linear amplification), which generates amplified RNA.
  7. The quality and concentration of the amplified RNA (aRNA) is determined by running the material on an Agilent Bioanalyzer system.
  8. Library preparation is performed to generate a sequenceable cDNA library.
  9. The quality and concentration of the cDNA library is 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. 

Updated on March 31, 2022

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