The transcriptome represents the entirety of all genes that are expressed, i.e. transcribed from DNA to RNA, in a cell at a distinct time point. Transcriptome sequencing (RNA sequencing, RNA seq) therefore allows the detection and quantification of all expressed RNA molecules in a biological sample. It is possible to carry out a targeted enrichment of only the mRNA molecules or to sequence total RNA exclusive rRNA and tRNA.
Application areas and objectives for exome sequencing in the scientific field are diverse and range from the analysis of differential gene expression to the detection of alternative splicing and new transcripts. Transcriptome sequencing therefore is carried out in basic research as well as in applied clinical research.
CeGaT specifically carries out mRNA sequencing and offers a comprehensive service from RNA extraction to data analysis.
Protocols may be easily adjusted to the requested application and the specific requirements of your samples. For example, we will adapt number and length of sequenced reads according to the customer’s needs. Upon request, we also offer a low-input protocol for samples with starting amounts as low as 1 ng of RNA.
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Please do not hesitate to contact us – we are happy to design an individual concept for your project.
If possible please provide us with information about sample material, number of samples and preferred sequencing depth.
When processing your sequencing order, we focus on delivering results with high quality and reliability. Each project is led by a scientific project manager, who will be your contact person during the entire project phase. Upon completion of your project, you will receive a detailed project report with further information regarding sample QC, important laboratory parameters and bioinformatic evaluation and explanations.
Preparation of sequencing libraries for samples with sufficient RNA content is carried out with the Illumina® TruSeq Stranded mRNA Library Prep Kit. This kit enables the comprehensive analysis of mRNA present in a certain sample and at the same time yields information about the strand orientation of the detected transcripts.
High-throughput sequencing is carried out on a Illumina platform.
Our standard sequencing depth is 30 M reads per sample. However, the sequencing depth can be individually adjusted according to the customers’ needs.
Depending on the goals and objectives of the transcriptome sequencing, we recommend different sequencing modes: 50 bp single end or 100 bp paired end. Possible combinations of sequencing mode and read numbers and their exemplary applications are shown in the table below:
30 M Reads
Custom-specific read number
50 bp single end
Differential gene expression, Detection of transcripts with average expression
Depending on the selected read number increase or decrease of detected transcripts. Higher read numbers can make sense for detection of weakly expressed transcripts or for the detection of tissue mosaicisms as they are found in tumors.
100 bp paired end
Detection of alternative splicing
Depending on the selected read number increase or decrease of detected transcripts.
Table 1: Combination possibilities offered for mRNA sequencing and their exemplary applications
For the evaluation of transcriptome data you can select between different levels of evaluation.
If desired, we are processing your data in a way that will allow you to directly start the functional evaluation of your data. This includes the calculation of expression values and the differential expression for experimental groups so that you receive information about expression values, up- and downregulated genes and a statement about changes in expression (“fold change”).
If you wish to perform additional analyses, we provide you with data for each intermediate step (raw data, aligned reads). Please note that for the calculation of statistically significant changes it is necessary to include a sufficient number of replicates per experimental group.
In case you wish to carry out the entire data analysis according to your own ideas and requirements, it is of course possible to simply receive the raw data of your sequences (FastQ format, demultiplexed, adapter trimmed).
On request, we also carry out further analyses such as de-novo transcriptome assembly, variant calling or annotation of variant lists for specific cases.
Our delivery includes the following services, which can be added step by step:
RAW: raw data in FastQ format (*fastq.gz)
Possible for all species
You will receive the sequencing data as it comes from the sequencer. Technical adapter sequences are already removed.
MAP: aligned reads in BAM format (*bam)
Possible for human, mouse, rat and C.elegans
Reads are aligned against the genome of the analyzed species using STAR
DE: Expression values and information about differential gene expression (*tsv)
Possible for human, mouse, rat and C.elegans
You receive a table with expression values and tables indicating up- or downregulated genes (fold change, pValue, qValue), respectively, between all pairs of defined experimental groups. Please note that a sufficient number of replicates per experimental group is required for the calculation of the statistical significance. If necessary we happily offer our advice in this respect. Moreover, you will receive a report about the top 10 differentially expressed genes and the top 10 of expressed genes. For a first estimate about the resemblance of expression profiles between your samples, we will carry out a hierarchic clustering analysis where you will receive the results as a figure.
Detailed project report (for all levels of analysis)
Our project report includes information about the results of sample quality controls, kits and protocols used in the laboratory, as well as the programs and versions used during data processing.
RNA: at least 1µg for the standard protocol; at least 1ng (at a concentration >50pg/µl) for the low input protocol, RIN ≥ 8.0. RNA must be shipped on dry ice.
Cells or tissue: at least 25mm3 or 5×105 cells for the standard protocol, at least 200 cells for the low input protocol. Tissue and cell pellets should be flash frozen in liquid nitrogen or dry ice and shipped on dry ice. Alternatively, it is possible to stabilize cells or tissue in „RNA later“ (available by different providers) and to ship the samples at room temperature.
Data transmission: Data will be delivered on a hard disk via mail.
Processing time: 6-8 weeks
Sample storage: The remaining DNA will be stored for 3 months after data delivery.