BioMicroCenter:Illumina Library Preparation
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'''RNA''' – Total RNA samples must be submitted in water or TE. The quantity of total RNA needs to be between 0.1-4ug
'''RNA''' – Total RNA samples must be submitted in water or TE. The quantity of total RNA needs to be between 0.1-4ug a RIN above 9.0.
Revision as of 15:49, 22 December 2011
Generating high-quality data on the Illumina sequencing platform requires high-quality libraries. The BMC currently offers library preparation services for genomic DNA and RNA-seq. Prior to sequencing, all samples must pass the BioMicro Center’s Sequencing Quality Control process, which verifies selection of inserts of a desired size and correct ligation of Illumina adapters.
Sample Prep questions can be directed to Michael Gravina and Ryan Sinapius.
Sample Prep Services
There are three options when submitting DNA for library prep:
BMC Fragmented DNA sample prep (SPRI and enrichment) - Samples will be pre-QC'd by BioAnalyzer, run on the SPRI-works system using BioMicro Center adapters (with size-selection at the user's option), enriched using BioMicro Center PCR primers (including molecular barcodes for multiplexing), final-QC'd, and either submitted for Illumina sequencing or returned to the user for their own sequencing.
BMC Fragmented DNA sample prep (SPRI-only) - Samples will be run on the SPRI-works system using user-supplied adapter mix and then returned to the user for their own enrichment. When choosing this option, please note that samples will need to be re-submitted if you plan to sequence with us later on.
BMC Intact DNA sample prep (NEXTERA) - Samples will be pre-QC'd by Nanodrop, processed using the Epicentre Nextera kit, enriched using Nextera PCR primers (including molecular barcodes if requested), final-QC'd on the BioAnalyzer and either submitted for Illumina sequencing or returned to the user for their own sequencing. At least 50ng of DNA is required for Nextera. Samples for Nextera cannot be pre-QC'ed on the BioAnalyzer because the large fragments will clog the microchannels.
There are three options when submitting RNA for library prep:
mRNA-seq - Samples will be pre-QC'd on the BioAnalyzer, poly-A purified and converted to cDNA using the Illumina Tru-Seq protocol, run on the SPRI-works system using BioMicro Center adapters, enriched using BioMicro Center PCR primers, and submitted for Illumina sequencing.
Strand Specific mRNA-seq - Samples will be pre-QC'd on the BioAnalyzer, poly-A purified and converted to cDNA using the dUTP 2nd strand marking protocol outlined in J. Levin et al 2010, run on the SPRI-works system using BioMicro Center adapters, enriched using BioMicro Center PCR primers, and submitted for Illumina sequencing.
Low Input mRNA-seq - Samples will be pre-QC'd on the BioAnalyzer, poly-A purified and converted to cDNA using the Ultra Low Input mRNAseq Kit from Clontech, run on the SPRI-works system using BioMicro Center adapters, enriched using BioMicro Center PCR primers, and submitted for Illumina sequencing.
ChIP-Seq: Sonicated chromatin will undergo IP on the IPstar. RNase A, Proteinase K, Phenol Chloroform extraction and purification will be performed off of the instrument and submitted for QC on the Bioanalyzer. These ChIP samples may be picked up for qPCR or sent directly to SPRI for library construction and sequencing (options are listed on submission forms). Users will be contacted if ChIP samples fail QC. These samples will not be submitted to SPRI-works but will be charged for the IP.
DNA– DNA samples must be submitted in either water or TE. Although the SPRIworks robot is not highly sensitive to the amount of DNA input, care must be taken to submit an appropriate amount of adapter to ensure efficient ligation. If you elect the Nextera kit instead of the SPRIworks, we will require 50ng of DNA input.
RNA – Total RNA samples must be submitted in water or TE. The quantity of total RNA needs to be between 0.1-4ug for Illumina TruSeq, 1ug-4ug for strand specific RNAseq, >100pg for Low Input RNAseq, all protocols require a RIN above 9.0.
Chromatin – Sonicated chromatin must be submitted at 5 million cells per 200uL volume. Antibodies must be submitted at the same time(at least 3ug of antibody per IP). We have done months of testing and highly recommend using the following sonication conditions:
1. Resuspend fixed cells in 10mL of LB1
2. Rock cells at 4 degrees for 10 minutes
3. Pellet cells at 4 degrees for 5 minutes at 1350g
4. Remove supernatant and resuspend cells in 10mL of LB2
5. Rock cells at 4 degrees for 10 minutes
6. Pellet cells at 4 degrees for 5 minutes at 1350g
7. Remove supernatant and resuspend cells in a volume of LB3 where there is 5 million cells/200uL
**Example: Resuspend 25 million cells in 1mL of LB3**
8. Sonicate cells
9. Pellet debris at 4 degrees for 10 minutes at 20,000g
10. Submit lysate for ChIP
Good quality control is crucial for optimizing the number of reads and quality of data produced by the sequencers. For more information on QC methods and protocols please visit the Sequencing Quality Control page.
- Fragmentation - All samples must be fragmented to the point where the majority of the fragment distribution is between 100 to 300 base pairs. RNA-seq samples are usually fragmented after poly-A selection and before first strand synthesis.
- End Repair and 3’ dA Addition – Any end damage that may have occurred during fragmentation is repaired and then an extra A is added to all 3’ ends to increase efficiency during ligation.
- Adapter ligation – Partial Illumina adapter sequences are ligated to the sample fragments, a total addition of 66bp between the two ends. Each adapter sequence has a T-overhang to increase efficiency. It is very important to maintain a good ratio of sample material to adapter. Illumina recommends a 1:10 ratio, to avoid the presence of primer-dimer after enrichment.
- Size Selection – This step is designed so that only the fragments within the desired size range (generally ~200–400bp) are included in the final library. Size selection is very important: fragments that are too large will create oversized, overlapping clusters on the Illumina flowcell and can result in a loss of read count in the final data. Fragments much smaller than the optimal size range may include residual primers and primer-dimers. Size selection is usually performed using an agarose gel.
- Enrichment – The DNA is PCR-amplified using the complete primer constructs required for binding and clustering on the flowcell. This adds 53bp of adapter sequence total between the two ends, for a final adapter length of 119bp. It is very important to optimize the number of PCR cycles so that there is sufficient material for clustering while limiting PCR biases.
Traditional Illumina protocols can be found on our protocols page.
Our BMC Fragmented DNA sample prep (SPRI and enrichment) service includes molecular barcoding to allow sequencing of multiple samples per lane. Nextera DNA prep and RNAseq-prep may include barcoding at the user's option.
Pricing and Priority
Full pricing information is available on our price list.