Sample Requirements

 

Sample preparation
We require that you send us the samples labeled with the codes provided after filling in the application form.

Shipping samples
DNA is shipped on regular ice while RNA has to be shipped on dry ice. Please make sure you use priority shipping and if possible send it in the beginning of the week to make sure the package won’t get stuck somewhere over the weekend!

 

Illumina
Library construction for single read and paired end seq (including samples intended for multiplexing)
Sample for preparing a DNA library starting from genomic DNA.

  • At least 1-5 µg of DNA.
  • OD 260/280 ratio of approximately 1.8.
  • Concentration of 50ng/µl or above in TE/EB.
  • Gel electrophoresis picture to verify that sample is high molecular weight.

Library construction for RNA-seq
Sample for preparing a cDNA library starting from total RNA or mRNA.

  • At least 1.5-10 µg of total RNA (or 0.1 µg of mRNA).
  • RIN or RIQ > 8.
  • OD 260/280 ratio of approximately 2.0.
  • Concentration of 50 ng/µl or above in TE or RNAse/DNAse free Water.
  • Result from Bioanalyzer.

Sequence Capture (in solution)
Sample for preparing a DNA library and perform sequence capture starting from genomic DNA.

  • At least 1-5 µg of DNA.
  • OD 260/280 ratio of approximately 1.8.
  • Concentration of 50 ng/µl or above in TE/EB.
  • Gel electrophoresis picture to verify that sample is high molecular weight.

 

Mate-pair library construction for PE seq
Sample for preparing a mate pair library starting from genomic DNA.

  • At least 5µg of DNA.
  • OD 260/280 ratio of approximately 1.8.
  • DNA should not be degraded (> 15 kb for a 2-5 kb span MP library).
  • Concentration of 200 ng/µl, in 10 mM Tris-HCl, pH 7.5-8.5, or Molecular Biology Grade Water.
  • Gel electrophoresis picture to verify that sample is high molecular weight. The majority of the DNA should run as a a high molecular weight band greater than 50kb in size and with minimal lower molecular weight smearing.

 

Library construction for ChIP seq

  • ChIP enriched sample, at least 200 ng in 30 µl water.

Libraries prepared by customer

  • The libraries must have a concentration of 10 nM.
  • Bioanalyzer analysis results must be added to the sample information.

The Genomics Core can not be held responsible for any data-loss related to the quality of the library.

 

GS-FLX
Library construction
Sample for preparing a Shotgun library through ligation of adaptors (performed on site)

  • At least 3-5 µg if DNA fragment size is larger than 1.5 kbp or close to this.
  • At least 1 µg if DNA fragment size is 70-1000 bp or close to this.
  • OD 260/280 ratio of approximately 1.8.
  • Concentration of 50 ng/µl or above in 10 mM Tris.
  • Gel electrophoresis picture and/or result from Bioanalyzer.

Sample for preparing a GS FLX+ Shotgun library through ligation of adaptors (performed on
site)

  • At least 1 µg of DNA with a fragment size larger than 2 kbp.
  • OD 260/280 ratio of approximately 1.8.
  • Concentration of 10 ng/µl or above in 10 mM Tris.
  • Result from Bioanalyzer.

Sample containing A and B adaptors (prepared by customer)

  • Suggested sample size range: 300-800 bp (diverse libraries), 100-800 bp (uniform libraries).
  • At least 10 ng/µl of DNA in 10 µl 10 mM Tris-HCl, pH 7.5-8.5, or Molecular Biology Grade Water.
  • Accurate concentration measurement.
  • Sample verified by gel or bioanalyzer.
  • The customer is responsible for removing short fragments from the sample.

The optimal size range to obtain high quality reads is 200-600 bp, including the Adaptor sequence. For amplicons shorter than 200 bp or longer than 600 bp the emulsion PCR protocols need to be modified.

If the experiment contains Amplicons of different sizes (e.g. range wider than 150 bp) this will reduce sequencing quality and should be avoided. One option would be to split the amplicon pools into subsets with narrower size ranges. Another option would be to pool amplicons at different ratios to counterbalance the effect of length on amplification efficiency. Though the optimal balance will have to be determined experimentally, a good starting point would be to add four times more of the longer Amplicons than the shorter ones.

 

Contact: genomicscore@uzleuven.be +32 16 33 08 21