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Next Generation Sequencing Core facility - Project submission

 

We recommend contacting the core facility to discuss the experimental strategy. New projects should be submitted by mail to the core facility via the Project Submission form. An official offer will then be posted by mail.

 

Investigators are kindly asked to follow the recommendations below. For any questions, please contact the facility.

General considerations

Sample quality will be checked at the facility to ensure optimal starting conditions before library synthesis. Investigators also have the option to send self-made libraries.

Genomic DNA

Purification of Genomic DNA can be done by phenol/chloroform extraction and ethanol precipitation or by column purification (e.g. Qiagen DNeasy Blood & Tissue Kit). Care must be taken to avoid shearing of the genomic DNA by pipetting and freezing/thawing. An example protocol for genomic DNA extraction can be found here.
Quantity requirements: Please provide 1 µg of purified genomic DNA in water or EDTA free buffer (for instance Qiagen EB buffer) and normalize the sample concentration to 50-100 ng/µl.
Quality requirements: Run an aliquot of the sample (approximately 50-100 ng) on a 1% agarose gel stained with ethidium bromide. The genomic DNA must appear as a single band of >10kb without lower molecular weight smear. The OD 260nm/280nm ratio must be ≥ 1.8.
Library fragmentation: Intact genomic DNA will be fragmented at the facility to a range of 200-400 bp on a Covaris Adaptive Focused Acoustics Model S220 instrument.

ChIP-Seq

Avoid carrier chromatin: Do not use ChIP methods involving the use of carrier chromatin as carrier DNA will also be sequenced.
Use low-binding tubes: The quantity of ChIP material being usually very limited, we highly recommend to use LoBind Eppendorf tubes for all steps of ChIP-seq experiments and storage.
Quantity requirements: Please provide 10 ng of ChIP DNA with a size of 100-200 bp at a concentration of 300pg/μl in water or EDTA free buffer (for instance Qiagen EB buffer).
Quality requirements: The OD 260nm/280nm ratio must be ≥ 1.8. A picture of the input DNA must be provided.

Exome Sequencing

Purification of genomic DNA can be done by phenol/chloroform extraction and ethanol precipitation or by column purification (e.g. Qiagen DNeasy Blood & Tissue Kit). Care must be taken to avoid shearing of the genomic DNA by pipetting and freezing/thawing. An example protocol for genomic DNA extraction can be found here.
Quantity requirements: Please provide at least 3 µg of purified genomic DNA. Samples can be resuspended in water or EDTA free buffer (for instance Qiagen EB buffer). Please normalize the concentration of samples to 50-100 ng/µl.
Quality requirements: Run an aliquot of the sample (approximately 50-100 ng) on a 1% agarose gel stained with ethidium bromide. The genomic DNA must appear as a single band >10kb with no lower molecular weight smear. The OD 260nm/280nm must be ≥ 1.8.
Library fragmentation: Intact genomic DNA will be fragmented at the facility to a range of 200-400 bp on a Covaris Adaptive Focused Acoustics Model S220 instrument.
Target enrichment: All Agilent SureSelect Target Enrichment kits for exon enrichment are available for human, mouse, zebrafish, dog and bovine genomes. Other custom made designs are possible. The lengthof capture oligos is 114-126 nucleotide with a coverage of 51,542,882 bases for the Agilent SureSelect Human All Exon v2 kit.

mRNAseq

Sample processing: It is critical to process biological samples as fast as possible in order to minimize RNA degradation. Rapid homogenization of freshly dissected tissue in Trizolreagent (Invitrogen) will ensure a high total RNA quality. We do not recommend storing dissected tissue before RNA extraction.
RNA purification: Extraction can be done with Trizol or by column based methods (e.g. Qiagen RNeasy kit). A protocol of total RNA preparation with the Trizol method can be found here. We recommend resuspending RNA in RNAse free water. Do not use custom-made DEPC-treated water.
Quantity requirements: Please provide 1 μg of total RNA at a concentration of 100-500 ng/μl. The minimum quantity to provide without amplification is 50 ng of total RNA.
Quality requirements: Total RNA must have an OD 260nm/280nm ratio of ≥ 1.8. Users should provide a picture of an agarose gel or Bioanalyzer (Agilent) profile showing no sign of degradation of the total RNA (RIN> 8).
Library preparation: mRNA will be purified from total RNA and processed for adaptor ligation (Illumina).

Strand-specific libraries: All RNA-Seq libraries are stranded (TrueSeq stranded RNA kit, Illumina).

Small RNAseq

Sample processing: It is critical to process biological samples as fast as possible in order to minimize RNA degradation. Rapid homogenization of freshly dissected tissue in Trizol reagent will ensure a high total RNA quality. We do not recommend storing dissected tissue before RNA extraction.
RNA purification: Samples must be provided as total RNA. Extraction can be done with the Trizol method, a protocol of which can be found here. If a column-based method is used make sure it is compatible with small RNA preparation. We recommend resuspending the RNA in RNAse free water. Do not use custom-made DEPC-treated water.
Quantity requirements: Please provide 2 μg of total RNA at a concentration of 100-500 ng/μl.
Quality requirements: The total RNA must have an OD 260nm/280nm ratio of ≥ 1.8. Users should provide a picture of an agarose gel or Bioanalyzer profile showing no sign of degradation of the total RNA (RIN> 8).
Library preparation: small RNAs will be size selected after adaptor ligation (target size 145-160bp).

rRNA depleted RNAseq

Sample to provide: We provide the rRNA depletion as part of the library preparation service for human, mouse, rat.
Sample processing: It is critical to process biological samples as fast as possible in order to minimize RNA degradation. Rapid homogenization of freshly dissected tissue in Trizol will ensure a high RNA quality. We do not recommend storing dissected tissue before RNA extraction.
RNA purification: Extraction of RNA can be done with Trizol or by column based methods (e.g. Qiagen RNeasy kit). A protocol of total RNA with the Trizol method can be found here. Samples must be provided as ribosomal depleted RNA. We suggest using the Ribominus (Invitrogen) or Ribo-Zero (Epicentre) kits. We recommend resuspending the RNA in RNAse free water (Ambion or equivalent). Do not use custom-made DEPC-treated water.
Quantity requirements: Please provide 100 ng of mRNA or rRNA depleted RNA at a concentration of 10-50 ng/μl, or 1 µg of total RNA if the depletion will be made by our facility.
Quality requirements: Total RNA must have an OD 260nm/280nm ratio of ≥ 1.8. Users should provide a picture of an agarose gel or Bioanalyzer profile showing no sign of degradation of the total RNA before rRNA depletion or polyA enrichment (RIN> 8). Please also provide a bioanalyzer profile after depletion or polyA enrichment.

 

 

 

 

Last modified: March 21, 2016