RNA-seq services

RNA-Seq Service Offerings

You can choose among three main RNA-seq service options - total RNA sequencing, mRNA sequencing, or miRNA/small ncRNA sequencing.

Total RNA

With total RNA-Seq services we will sequence the whole transcriptome which includes coding and non-coding RNAs. Our solutions include ribosomal RNA depletion. This approach is optimal for finding biomarkers from known transcripts and new RNA features.

mRNA

Sequencing of messenger RNA-Seq allows accurate estimation of transcript abundance for differential expression analysis as well as the study of alternative splicing and discovery of new transcript isoforms.

miRNA and small non-coding RNAs

Gene silencing and post-transcriptional regulation of gene expression can be easily investigated through our miRNA-Seq service.

1. Sample QC

RNA quality control: Measurement of concentration as well as determination of RIN value (RNA integration number) Note: RNA should be isolated in a high-quality manner. Qiagen portfolio kits for RNA extraction and purification are recommended. For sample preparation and sample shipment, please follow Diagenode’s guidelines, which will be communicated at the time of order. If samples do not meet quality requirements, additional QC on new samples may incur additonal fees.

Required quantities:

Three biological replicates are recommended.

• miRNA and small RNA-seq services: minimum 10 ng of total RNA.
• mRNA-seq services: minimum 10 ng of total RNA.
• Total RNA-seq - Long non-coding RNA and whole transcriptome sequencing services: minimum 5 ng of total RNA (Human, Mouse, Rat) or 100ng (Plant).

2. Library preparation:

• Poly-A selection for mRNA-seq or rRNA depletion for total RNA-seq (with lncRNA)
• Stranded library preparation technology
• Final QC prior to sequencing (quantification and fragment size)

3. Sequencing with Illumina technology

Single-End: 50 bp for standard studies in human, mouse or rat samples.

Paired-End: 75 bp for specific studies like splicing variant and rare transcript discovery in human,mouse, rat or novel species (unknown or poorly characterized transcriptomes).

• miRNA and small RNA-seq:10-20 million raw reads per sample.
• mRNA-seq: 25-30 million raw reads per sample.
• Total RNA-seq: 50-100 million raw reads per sample

4. Data Analysis

• Bioinformatic analysis: read alignment to genome, transcriptome or miRNome, abundance estimation, study of alternative splicing
• Statistical analysis: Differential expression analysis, samples clustering (k-means clustering and Principal Component Analysis)
• Enrichment analysis: gene ontology enrichment analysis and pathway enrichment analysis
• Data mining: biomarker discovery through machine learning algorithms Integration of data with other types of epigenomic datasets (ChIP-Seq, RRBS, ATAC-Seq, etc.)
• Data delivery of raw data and all generated files through fast and secure file transfer or hard drive upon request

Bioinformatics analysis options

Included in all services:

Alignment to reference genome and quantification of known genes and/or transcripts Provided files: Report with sequencing statistics, raw data in FASTQ format, FastQC report, Alignment in BAM format, Un-normalized counts, DESeq2 normalized counts matrix.

Files will be available for download for 1 month and stored for an additional period of 3 months on Diagenode’s servers. Additional long-term storage of data is available upon request. This offer includes a one hour call to walk you through the results if needed.

Additional bioinformatics analysis on request

• Differential expression: differential expression testing between samples for genes and/or transcripts.
• Gene ontology terms analysis: analysis of differentially expressed gene sets. Gene Ontology terms that are overrepresented in differentially expressed genes may indicate the underlying biological processes involved.
• Pathway analysis: identification of biochemical pathways in which genes associated with differential expression may be overrepresented.

Original samples are stored at Diagenode during 12 months after project completion, but will be discarded once this time is exceeded. Return shipment of samples is available upon request.