U of L Sequencing Technology Center
Illumina Next-Generation Sequencing
We operate a full suite of standard Illumina NGS preparation and sequencing pipelines, including both DNA and RNA sequencing. Libraries are processed using the most cost-effective sequencing solution based on the project needs and batch sizes, using either the MiSeq or NextSeq 2000 systems. We also accept pre-made libraries for sequencing.
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Listed below are our most requested applications and an overview of general input requirements.
Do not hesitate to contact us if you are interested in working with a method not listed below.
Illumina Stranded mRNA
RNA-seq library preparation method to specifically investigate mRNAs. Polyadenylated RNA molecules are enriched via priming using oligo-dT, and strand information is preserved. Total RNA must be DNAase treated prior to submission. Not FFPE compatible.
Input requirements: total DNAse-treated RNA with RIN >7; 500ng minimum at a concentration of 15-20 ng/uL resuspended in nuclease-free water.
Small RNA
Library preparation method used to study small non-coding RNAs (including miRNAs) responsible for gene silencing and post-transcriptional regulation of gene expression, using the NEB Next small RNA protocol.
Input requirements : 200 ng of DNAse-treated total RNA (including small RNAs) with RIN > 7 at 20 ng/uL OR purified small RNAs from 200ng DNAse-treated total RNA, resuspended in nuclease-free water.
Illumina DNA Prep
Double-stranded DNA preparation method used for next generation sequencing of whole genomes . Method optimized for a wide range of input from 1-500 ng DNA for small genomes (e.g. microbial) and 100-500 ng for large genomes (e.g. human).
Input requirements: 300ng of intact gDNA at 20 ng/uL in water, EB or low TE (<0.1mL EDTA)
Illumina Stranded Total RNA
RNA-seq library preparation that enables study of whole transcriptomes including protein coding mRNA and non-coding RNAs, while preserving strand information. The method is compatible with low quality, degraded and FFPE RNA samples. The kit specifically depletes abundant cytoplasmic RNA species (i.e., rRNA, mtRNA, globin RNA) from human, mouse and rat origin. Alternative methods are available for microbial and non-model species
Input requirements: 500ng of DNAse-treated total RNA at 15-20 ng/uL resuspended in nuclease-free water.
16S rRNA profiling
Targeted amplification of the V3-V4 region of the bacterial 16S rRNA followed by NGS for taxonomic profiling of bacterial communities. Up to 96 samples can be analyzed per pool using the MiSeq system. Please contact us for recommendations on DNA extraction methods to limit host contamination and enhance on-target amplification.
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Input requirements : Input requirements and downstream protocol (performed by the ST team) are detailed in this white paper.
Amplicon Sequencing
Nucleotide-level resolution of small amplicons (up to 500bp) to identify small variants or validate mutagenesis strategies. Multiple preparation strategies available based on sample number and scientific question. Please contact us for experimental design consultation to outline specifications.
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Input requirements: 250ng of intact gDNA at 20 ng/uL in water, EB or low TE (<0.1mL EDTA)