RNA Sequencing

RNA Sequencing (RNA-Seq) harness the capabilities of high throughput sequencing method to have better understanding of transcriptome of cells. The sequencing data will facilitate the discoveries of novel transcript, identification of alternatively spliced genes, and detection of allele-specific expression. Eurofins offers end-to-end solution of RNA-Seq of all the biological samples using Illumina, Pacbio platform. We have completed various RNA-Seq projects and delivered data to our clients. Our expert team will help to design your work flow based on objective of project/study.

Depending on your needs there are three different services available for transcriptome sequencing projects. A specific set of libraries is available for each section, offering data generation according to your needs.

• De Novo and Reference based RNA Seq
• Small RNA-Seq

De Novo RNA Seq

Identify the genes present in your transcriptome. De novo sequencing of transcriptomes: What is possible? De novo transcriptome sequencing analyses expressed genes present at a specific time and with a specific physiological background. With the average transcript length of 1,500-2,000 bp, several reads have to be generated per transcript, which are later assembled to reconstruct the full length transcript. Sequencing our random primed cDNA library on Illumina NextSeq 500 or HiSeq 2500, Pacbio provides you with tens of millions of 75 bp, 100 bp or 150 bp reads. Usually the output of one channel is way too high for one transcriptome, meaning that several libraries can be analyzed in one channel. To your convenience, Eurofins has built up a proprietary software pipeline for de novo assembly of short Illumina reads to cost-efficiently reconstruct the transcripts.

Advantages:

• Detect rare or previously unknown transcripts
• Strand specific protocol (upon request)
• Cost-efficient de novo transcriptome data

Reference based RNA Seq:

Analyze gene expression responses and rely on our long-term experience: mRNA-Seq for gene expression analysis and detection of splicing events. Gain one of the most comprehensive views of all expressed transcripts within your sample with our RNA-Seq services. Since the entire transcriptome is covered with short reads, RNA-Seq is the preferred method to analyze novel transcripts, novel isoforms, alternative splice sites, rare transcripts or SNPs.

Advantages:

• Analyze gene expression, gene expression abundance, alternative splice sites and SNPs.
• Strand-specific protocol (upon request)
• Obtain comprehensive view of the entire transcriptome
• Analyze prokaryotic transcripts by rRNA depletion
  
  
  

Methodology (for De novo and reference based RNA sequencing)

We accept isolated pure RNA, microbial cultures (non‐pathogenic), animal/plant tissues, etc

Sample requirement:

• RNA: Submit DNase treated RNA of approximately 3-5µg of high quality with Nanodrop A_260/280 ratio >1.9; A_260/230=2.0-2.2; and at a concentration of ~100 ng/µl (dissolve in nuclease free water). Supplied RNA should be shipped in dry ice to the Eurofins facility.
  • Plant/Human/Animal Tissue: Tissues of 2-4 gm should be provided in RNA later or snap frozen tissue without any storage solution shipped in dry ice.
  • Microbial non-pathogenic culture: Snap frozen pure isolated culture pellet (Bacteria / Yeast / Fungi) should be shipped in dry ice.

Sample Quality Control:

• NGS grade High quality RNA will be isolated from the supplied sample using commercially available kits.
• The qualification and quantification of RNA will be carried out by denatured agarose gel electrophoresis and RNA profiler/ NanoDrop/Qubit Fluorometer analysis.
• RIN value estimation will be carried out using Agilent 4200 Tape Station.

Library Preparation:

• RNA Seq library will be prepared by using TruSeq Stranded mRNA Sample Prep Kit or TruSeq Stranded Total RNA Library Prep Kit (depending on the requirement).
• Library validation will be carried out using Agilent 4200 Tape Station.

Sequencing:

• Sequencing will be carried out on Illumina NextSeq500 platforms with 2 x 150 bp v2 chemistry or HiSeq 2500 with 2 x125bp v4 chemistry or NovaSeq6000 with 2 x 150bo chemistry.

Deliverable:

• Raw data will be available for download as a compressed archive of FASTQ files for each sample.
• Comprehensive compiled report and data set will be shared in link or Pen Drive or Hard Disk.
• For analysis please visit Bioinformatics Services

Turnaround time

5-7 weeks after clearance of QC process. It depends upon data size, scope, technology selected, number of samples and complexity of the project. The TAT is offered expecting no biological or technical difficulties for processing of all project samples

Small RNA Sequencing

Discover new small RNAs and analyse the expression of known PTGS process.

Small RNAs constitute a family of RNA species that plays a central role in regulating silencing and post-transcriptional gene expression regulation processes in many organisms. With the enormous depth reached by next generation sequencing technologies, you can get a comprehensive view of the small RNAs in your samples would be essential part in the identification of novel marker.

Sequencing small RNA is done by preparing a short insert cDNA library for each sample and subsequently sequencing these libraries using Illumina NextSeq 500 or MiSeq technology using single end 1x75 a minimum of 10million read per sample. To sequence several small RNA samples in one channel, we offer library preparation with individual indexes. Eurofin offers comprehensive small RNA sequencing and analysis (sRNA-Seq) to investigate the regulatory role of non-coding RNA of 18-40bp in length

Typical projects application are:

• Differential expression analysis of small RNAs
• Identification of novel small RNAs
• Identification of target molecules of novel miRNAs
• Functional verification, knockout or over expression of miRNA genes

Methodology

We accept pure RNA, microbial cultures (non‐pathogenic), animal/plant tissues, etc

Sample requirement:

• RNA: Submit DNase treated RNA of approximately 3-5 µg of high quality with Nanodrop A_260/280 ratio >1.9; A_260/230=2.0-2.2; and at a concentration of ~100 ng/µl (dissolve in nuclease free water). Supplied RNA should be shipped in dry ice to the Eurofins facility.
  • Plant/Human/Animal/ Insect Tissue: plant Tissues of 2-4 gm, Animal tissue (50-100mg) should be provided in RNA later or snap frozen tissue without any storage solution shipped in dry ice to Eurofins.
  • Microbial non-pathogenic culture: Snap frozen pure isolated culture (Bacteria / Yeast / Fungi) should be shipped in dry ice.

Sample Quality Control:

• NGS grade High quality RNA will be isolated from the supplied sample using commercially available kits.
• The qualification and quantification of RNA will be carried out by denatured agarose gel electrophoresis and NanoDrop/Qubit Fluorometer analysis.
• RIN value estimation will be carried out using Agilent 4200 Tape Station.

Library Preparation:

• Small RNA Seq library will be prepared by using TruSeq small RNA library preparation Kit.
• Library validation will be carried out using Agilent 4200 Tape Station.

Sequencing:

• Sequencing will be carried out on Illumina NextSeq500 platforms with 1 x 75 single end chemistry or on NovaSeq platform with 1 X 50bp chemistry.

Deliverables:

• Raw data will be available for download as a compressed archive of FASTQ files for each sample.
• Comprehensive compiled report and data set will be shared in link or Pen Drive or Hard Disk.
• For analysis please visit Bioinformatics Services

Turnaround time

Provided that there are no unexpected difficulties (biological or technical), 6-8 weeks after successful clearance of QC step. It depends upon data size, scope, technology selected, number of samples and complexity of the project.