Preparation of DNA/RNA libraries for Next Generation Sequencing projects
In recent years, the demand for Next Generation Sequencing (NGS) methods in stem cell research has increased rapidly. However, the technical challenge of library production is often daunting and time-consuming, and forms the major bottleneck of many projects. To accommodate this, the Wellcome Trust Centre for Stem Cell Research has created a facility which will produce your NGS libraries on demand.
This facility provides a fast turnaround for standard library preparation. It provides a local and flexible service to anybody within the Cambridge Stem Cell Institute.
- Project planning and customisation
- Covaris shearing of DNA/RNA samples; chromatin samples
- Transcriptome/mRNA libraries
- Small RNA libraries
- ChIP-seq libraries
- Genomic resequencing libraries
- WGBS projects
- Exome sequencing
- Individual projects and non-established methods will be considered
Currently available preparations/kits
- Illumina small RNA library kit
- Nextflex directional RNA seq kit
- Nextflex rapid DNA kit
- Nextflex adapters for multiplexing up to 36 samples
- Thruplex DNA-seq kit
- NEBNext reagents for standard Illumina DNA libraries and ChIP libraries
- NuGen Ovation Mouse RNA-Seq Library System
- NImbleGen SeqCap EZ exome library kits
- Smartseq2 method for single cells/low RNA amounts
- Covaris E220 evolution instrument for DNA/RNA shearing and chromatin shearing
The sequencing itself is not done in-house, but we have a collaboration with the CIGC here in Cambridge which allows us acess to the latest Illumina HiSeq and MiSeq sequencing technology.
If you are using the NGS library facility, the sequencing will be submitted directly to sequencing. If you are making your own libraries and would like access to the sequencing core, your group will need to set up an account with CIGC, and submit the SLX submission files for approval to Maike Paramor (firstname.lastname@example.org).
The SCI has a bioinformatics facility, which will provide support with any aspect of your data analysis.
The Gene Service Facility is managed by Dr Maike Paramor.
The Academic chair for this facility is Dr Michaela Frye.
How to book
Please find the sample submission form which you can download and fill in.
When submitting samples, please follow these simple guidelines:
Always make sure you have got the best possible quality of your RNA or DNA - to get good libraries, you need good starting material! Quantify your nucelic acid with a fluorescent dye (Qubit, picogreen), and avoid the nanodrop!
Total RNA for standard RNA-seq projects (2-5ug) will have to be DNAse digested and submitted in RNAse free water, the volume should not exceed 30 ul. Please check the quality of your RNA if you can, and aim for bionalayzer RIN values for >8. We can do this quality check for you on request, should you not have access to a bioanalyzer.
If quantity of total RNA is limited, please discuss options with me. The volume needs to be considerably smaller for low input samples.
Genomic DNA should be free of RNA, and you should check the integrity and size on a 1% gel before submitting.
Clean ChIP DNA should be submitted after you have done the chromatin immunoprecipitation in a volume not larger than 35ul. The minimum amount of DNA should not be less than 3ng. Do not attempt to measure ChIP DNA in a nanodrop, it will not be accurate enough. Please let me know if your fragments are longer than 500bp.
To make a booking, please go to the online Booking System.