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Genomic and RNA Profiling Core Facility

Houston, Texas

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Genomic and RNA Profiling Core
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Covaris Shearing Services

GARP offers sample sonication as a service using the Covaris S2 instrument. This instrument uses Adaptive Focused Acoustics (AFA) to precisely deliver energy to a specific area within a sample tube. Using AFA tubes, this technology accurately fragments DNA and RNA to the 100-1500 bp size range. The samples are sheared in isothermal conditions in a closed vessel to avoid degradation, cross-contamination or sample loss.

Covaris DNA/RNA Shearing for NGS

DNA Shearing
DNA Input 100 ng to 5 ug of purified gDNA or cDNA
DNA Quality
  • Genomic DNA should be intact (>10 kb).
  • cDNA size ranges will vary.
  • A gel (gDNA) or bioanalyzer trace (cDNA) should be used to assess degree of degradation.
Recommended Buffer

1X Tris EDTA (provided the EDTA does not cause any interference in downstream applications).

Water is NOT recommended.

Fragment Size Range

(target bp peak)

150 to 1,500 bp
To submit
  • Follow instructions found in the Sample Submission tab to enter an SAQC request and submit samples.
  • In the notes section of the request, indicate that the samples are for shearing and specify the desired target size.

RNA Shearing
RNA Input

mRNA : 1 μg

Total RNA: 5 μg

RNA Quality

RNA quality should be assessed to determine if any degradation is present in the sample, as degradation will alter shearing conditions.

Recommended Buffer

1X Tris EDTA, pH 8, or 10mM Tris-HCl, pH 8.5

Sample Volume 130 μl

Fragment Size Range

(target bp peak)

200 bases. This will vary based on the shear time. Optimizing shear time for each sample type is recommended.
To submit
  • Follow instructions found in the Sample Submission tab to enter an SAQC request and submit samples.
  • In the notes section of the request, indicate that the samples are for shearing.

Click here for more information on Covaris DNA/RNA shearing for NGS.

Covaris Chromatin Shearing

Covaris offers two truChIP™ Chromatin Shearing Kits: truChIP™ Chomatin Shearing Reagent Kit for mammalian cell lines and truChIP™ Tissue Chromatin Shearing Kit with SDS Shearing Buffer. Please follow the manufacturer’s instructions for these kits (see NOTES below).

Chromatin Shearing
Cell Number Input

1-30 x 106 cells

Sample Volume

Low Cell protocol (1-3 x 106 cells) – 130 μL

High Cell protocol (5-30 x 106 cells) – 1 mL

Shear Time

Low Cell protocol – 2 to 12 minutes.

High Cell protocol – 2 to 20 minutes.

Optimization of shear times should be conducted whenever experimental parameters change (e.g. cell type, cell number or sample volumes).

To submit
  • Follow the instructions found in the Sample Submission tab to enter an SAQC request and submit samples.
  • In the notes section of the request, indicate that the samples are for shearing.
  • Due to the nature of the protocol, shearing must be done the same day as nuclei isolation (see notes below). Before processing samples, contact GARP to confirm availability of the Covaris instrument and GARP technicians.
Notes
  • Fixed cells can be stored at -80oC for 2-3 days. Longer storage times may negatively affect shearing and reproducibility.
  • Isolated nuclei should be sheared immediately and not stored, as storing can negatively affect IP efficiency.
  • Sheared chromatin can be stored at 4oC for up to 2 days. Freeze/thaw cycles can dramatically reduce IP efficiency and reproducibility.

Click here for more information on Covaris Chromatin shearing.

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