Sequencing Facility Sample Preparation and Submission

Sample Preparation and Submission

The sequencing facility at CRI is open to all scientists at CRI, UT Southwestern and other interested institutions. For access to the sequencing facility, please contact Yoon Jung Kim or Sihan Wu at least a week before you plan to run sequencing.

Prepare sequencing libraries

We suggest you pool the bar-coded samples if you are using multiplexed libraries.

For help with this, please see the workflow overview below:

  1. Prepare samples (RNA, ChIP DNA, genomic DNA, etc.)
  2. Choose a library preparation kit
  3. Prepare the library
  4. Complete library QC
  5. Library pooling (if using multiplexed library)

Choose a library preparation kit. You can obtain library prep kits from several vendors (Illumina and NEB). Once you have decided which kit to use for library prep, please follow the protocols the manufacturer provides. Pay attention to the minimum amount of DNA or RNA required for each kit and whether the provided indexing (or bar-coded adaptor) system is compatible with Illumina sequencing technology.

The recommended library prep kits for RNA-seq or ChIP-seq samples based on our experience.

Check the quality of your samples

Proper sample quantification is extremely important. Please follow the “NGS Library Dilution and QC Analysis” protocol for this important step. Complete the Library QC Form , and email it to Yoon Jung Kim or Sihan Wu , along with the Sample Drop-off Form.

Provide pooled library and complete the necessary forms.

Provide pooled library as the following: 10 nM, 20 ul is recommended but the minimum amount is 4 nM, 20 ul.

Email the Sample Drop-off Form and Library QC Form to Yoon Jung Kim or Sihan Wu.

Submit the pooled library.

Send the pooled library to Yoon Jung Kim or Sihan Wu in room NL12.110L.

Please note the minimum sequencing unit is one flow cell (300~400 million reads, which is equal to the reads from two lanes in an 8-lane flow cell used in most facilities). We do not accept libraries with 5′ inline bar codes, because Illumina sequencing is sensitive to constant sequence within the first few bases of the read. We use Illumina NextSeq 500 High Output kits (#FC-404-2002; FC-404-2004; FC-404-2005) and Medium Output kits (#FC-404-2001; FC-404-2003) to perform sequencing. Please make sure that your library preparation method is compatible with Illumina sequencing primers.