Processing

Processing

This article explains what you can expect from processing your bulk RNA sequencing samples.

Requirements

In short, our requirements to start processing are:

  • Samples are submitted online
  • A printed version of your sample submission is added to the package
  • You have signed a quotation

Before processing, we check if we have received your sample submission form online and if your printed sample submission form is added to the package. The info on the sample submission form is required for correct processing in our lab. Besides submitting the samples, we also check if you have a signed quotation

We do not process samples that do not have a completely filled-out submission form and/or do not have a signed quotation.

Throughput time

We maintain a throughput time of 4 to 6 weeks. We start counting from the moment your samples arrive. In these weeks we process your sample in our lab, send it out for sequencing, perform our preliminary data analysis and sent it back to you.

Workflow

When bulk-seq 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.

*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. 

Lab updates

You will receive two or three updates by email, depending on the samples you submit. The first one is to inform you about the start of processing your bulk-seq samples. After the first update, it will take a maximum of two weeks before you receive the second update.

The second update is about the preliminary QC results of the quality of the total RNA. You will only receive this email update your submitted samples contained high enough cell counts per sample, as this QC check can only be done if samples have enough material to run on the bioanalyzer before processing.

The third update is the QC data results for aRNA and library cDNA. In this email, we explain how we interpreted the QC data and what the results are. The email will also contain 2 PDF files with aRNA and cDNA QC data (bioanalyzer plots), so you can have a look for yourself as well.

In this email, we will also tell you if we think it is possible to sequence your sample. If your sample is good enough to be sent for sequencing, we will send it out without consulting you first. If the QC data of your sample is questionable, we will consult you first about if you want to proceed with these samples.

QC

Bioanalyzer plots

During the processing of bulk-seq samples, we check the amount of aRNA and cDNA and the quality of the samples. The plot it generates is a QC metric to study the concentration and size distribution of library fragments and is the first indication of the quality of the sample.

The QC results depend e.g. on the cell type, number of cells per sample, and the quality of the extracted material.

QC workflow

After your samples arrive at our lab, we either perform TRIzol extractions and/or RNA concentration normalization of all your samples, depending on the type of samples you send us (cells in TRIzol vs extracted RNA) and whether or not there is enough starting material for concentration normalization. Next, we perform a reverse transcription and second strand synthesis reaction. Subsequently, the material from all samples is pooled into a single Eppendorf tube to perform In Vitro Transcription (IVT); a linear amplification step that results in amplified RNA (aRNA).

We fragment this aRNA and then run it on an Agilent bioanalyzer. If possible, based on the number of cells provided per sample, we also check the quality of the RNA before amplification. However, for some cell types this may not be possible with samples containing fewer than ~10k cells each.

After IVT, we perform another reverse transcription reaction as well as a PCR reaction. During the PCR reaction, the material is again amplified and the right adapters for sequencing are added to the sequences. This results in a DNA library (cDNA) that can be used to send for sequencing.

We run the cleaned cDNA library on an Agilent bioanalyzer.

Total RNA plot

To check the quality of the input material, we run your samples on the bioanalyzer. In every total RNA plot you first see a marker peak at 25 nt. For mammalian samples, there should be two very distinct peaks at 1500-2000nt and 3000-5000nt approximately. These are the ribosomal 18S and 28S peaks. Peaks between the marker peak and the ribosomal peaks indicate degraded material.

The ratio of the two ribosomal peaks to the total area under the curve determines the RIN value. The RIN can have a value from 1 to 10, with RIN=10 being the least degraded RNA and RIN=1 being completely degraded. We recommend a value of ≥6 (for mammalian samples).

aRNA plot

In every aRNA plot, you first see a peak at 25 nucleotides (nt). This is a marker peak. What should follow is a distinct RNA “bump”.

Depending on the cell type, the quality of the cells, the number of cells, and the quality of the reaction, the amount of RNA can vary significantly. A low RNA yield is in some situations very normal (think: low RNA expressing cells, RNA from nuclei, etc.), while in other situations it may indicate a problem with for example the number or the quality of the cells.

A small “extra bump” may precede the RNA curve. This is a primer-dimer peak and does not interfere with the quality of your sample.

Also important to mention is the RIN number: this number usually says something about the quality of your RNA and is calculated based on the ribosomal peaks of your total RNA. Since here we do not measure total RNA and we fragment the aRNA before running it on the bioanalyzer, the RIN number has no meaning anymore and can be neglected.

cDNA plot

In every cDNA plot, there are two marker peaks: one at 35bp and one at 10380 bp. There should be a clear and spiky curve between these markers, that goes up at around 200 bp and is back at baseline around 2,000 bp.

The height of the curve depends mostly on the number of PCR cycles but can be limited by a low RNA yield (a very low to absent RNA yield, which will usually result in a low(er) DNA curve even at the maximal cycle number).

Sequencing

Updated on July 20, 2021

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