Bulk Techniques

We can prepare the following RNAseq libraries:

mRNAseq: Profiling of the coding mRNA transcriptome. From total RNA, poly-A –captured RNA libraries are prepared and differential gene expression analysis can be performed. Sequencing is performed on NovaSeq6000 52bp paired-end, aiming for 30M reads per sample. 

Total RNAseq: A whole transcriptomic technique. Total RNA is rRNA depleted and reverse transcripted to cDNA. After amplification and sequencing, the whole transcriptome (mRNA and long noncoding RNA) can be assessed with strand specific information of the transcripts. Sequencing is performed on NovaSeq6000 52bp paired-end, aiming for 30M reads per sample. 

Small RNAseq: Small RNA transcriptomic analysis. From total RNA, libraries are prepared and differential analysis of small RNAs such as miRNA can be assessed. Sequencing is performed on NextSeq500 38bp paired-end, aiming for 5M reads per sample.

A reduced representation of the whole genome methylation patterns in a single nucleotide resolution. By restriction enzyme cleavage, the DNA becomes enriched of CG- fragments. After adapter ligation and sample pooling, the DNA is bisulfite converted and libraries are amplified. A differential methylation analysis can be performed. Sequencing is performed on NovaSeq6000 100bp single-end, aiming for 60M reads per sample.

Whole genome bisulfite sequencing is a technique used to detect genome-wide methylation patterns in a single nucleotide resolution. Extracted DNA is sheared following adapter ligation. To distinguish unmethylated and methylated cytosines, the libraries are chemically modified by sodium bisulfite. After library amplification, a quality check is performed and the libraries are sequenced on the NovaSeq.

SCOP can provide detailed protocols for:

• Chromatin ImmunoPrecipitation (ChIP)-sequencing
• Genome-wide Chromatin Conformation Capture (HiC)- sequencing
• Assay for Transposase-Accessible Chromatin (ATAC)- sequencing

The isolation of DNA / RNA is not included in SCOP’s services therefore:

Make sure to see SCOP’s isolation recommendations and requirements below. Good quality is important as low quality or degraded samples will affect the data.

RNA sequencing (mRNA or total)

• Isolate the RNA by column-based method (can be combined with Trizol).
• Treat with DNase during isolation protocol to ensure pure RNA.
• Measure concentration and define purity using a NanoDrop. Ratio: OD260/280 ~0.
• Measure quality using a capillary electrophoresis method (Bioanalyser / Tapestation). RNA integrity number (RIN)must be > 0.
• Recommended quantity 250ng.
• For total RNA, the recommended concentration is >25ng/µl.

Small non-coding RNA sequencing

• Isolate the RNA by column-based method (can be combined with Trizol). Ensure to capture small RNA.
• Treat with DNase during isolation protocol to ensure pure RNA.
• Measure concentration and define purity using a NanoDrop. Ratio: OD260/280 ~0.
• Measure quality using a capillary electrophoresis method (Bioanalyser / Tapestation). RNA integrity number (RIN)must be > 0.
• Recommended concentration >20ng/µl (if less, please contact SCOP).
• Minimum quantity 200ng.

Reduced Representative Bisulfite Sequencing (RRBS)

• Isolated the DNA by a column-based method.
• Measure concentration using Qubit.
• Define purity using a NanoDrop. Ratio: OD260/280 ~1.8.
• Minimum concentration 12 ng/µl
• Minimum quantity 100 ng (as measured by Qubit).

Please prepare samples for SCOP as follows:

• Randomize samples (please take the experimental setup into consideration upon randomization)
• Dilute samples and prepare them in 0.2ml strips with orientation as in columns in numerical order. Input concentration and volume differ between assays, see below for required total volume per sample.
• If SCOP is performing RNA QC, then prepare 10µl of RNA (conc. 1-2ng/µl) in 0.2ml strips.
• The samples should be numbered from 1 and onwards and marked on lid and tube side.
• Deliver samples in a box labeled with your SCOP project ID.

Required sample concentration and volume:

The three main experimental steps are:

1. Sample drop-off

RNA/DNA should be isolated and prepared by user using the recommendations for sample preparation (see table under Sample preparation and requirements). Samples are delivered to SCOP.

2. Library preparation

• Library preparation will be performed.
• QC of final RNA-sequencing library (Tapestation) will be performed, the concentration of library will be measured to determine the ratio for pooling prior to RNA-sequencing.

3. Sequencing

Samples will be pooled and sequenced on an Illumina NextSeq or NovaSeq depending on the number of samples and application. This normally results in a library excess, which will be stored for potential additional sequencing if requested by the user or if the initial run fails. All material (leftover cDNA/DNA and library) may be picked up by the user within one month of sequencing.

The data processing and analysis may include any combination of the following steps:

• QC of sequencing results.
• Read alignment to the reference genome.
• Differential expression or Methylation analysis.
• Ontology enrichment analysis using different databases (e.g. GO, KEGG, REACTOME) and methods (e.g. ORA, GSEA, CAMERA, FRY).
• Rhythmicity/Differential rhythmicity analysis for circadian clock experiments.

1. Data and results handover. Formats and examples of results.
2. Consideration related to data sharing upon publication (upload the GEO).
3. Storage of raw data.
4. User feedback.

Users of SCOP's NGS services are charged for the reagents used for library preparation and sequencing. SCOP covers all expenses associated with work time and currently SCOP is offering a 30% discount on the total price for all CBMR users.

Users of SCOP's services are charged for the reagents used for library preparation and sequencing. SCOP covers all expenses associated with work time, and currently SCOP is offering a 30% discount on the total price for all CBMR users.

The price for services depends on the library preparation and sequencing cost. The sequencing price per sample usually becomes cheaper as the number of samples increases.

See below an overview of the library preparation cost, as well as some price examples for the different library preparation services we offer. If you would like us to provide a cost estimate, then please contact us with information about the number of samples and techniques you would like us to run.

Library preparation cost, per sample:

Total RNAseq – DKK 575
mRNAseq – DKK 360
small RNAseq – DKK 500
RRBS – DKK 530
WGBS – DKK 930

You can download an overview of SCOP's pricing for Bulk NGS here.

Please recognize and acknowledge SCOP’s contributions to the scientific improvements in your project. Being acknowledged in publications is very important as it serves as documentation for the SCOPS's value and performance.

For a guide on how to acknowledge SCOP, click here.