Next Gen Sequencing Library Preparation
Next Gen Sequencing Library Prep
Core customers can either prepare their own libraries using unique or experimental protocols or use our existing libraries prep services. The core can work with you to identify or devise a library construction method that fits your needs.
The core offers a range of standard NGS library prep protocols and can develop new protocols to suit customer needs. Standard library preps can be requested using the Illumina Next Gen Sequencing Project Request in iLab (the same request as is used for requesting Illumina sequencing runs on the NextSeq and MiSeq). Please direct all new inquires to Dr. Katia Sol-Church for consultation.
For all RNA libraries the input total RNA is checked for concentration and quality using the NanoVue and Agilent Tape Station. Required concentrations are listed for each library prep type. All protocols recommend a RIN value of at least 7 (determined using the Tape Station), but we will run lower quality samples at the user’s request.
NGS RNA-seq of mRNA
(see iLab service: NGS library prep – RNAseq – M)
NGS RNA-seq libraries for the sequencing of mRNA are prepared using the NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina® with NEBNext® Poly(A) mRNA Magnetic Isolation Module. Starting material is 100 ng–1 μg of total RNA, which will be diluted to a volume of 50 μl. Please provide additional material for QC checking with NanoVue and TapeStation.
NGS RNA-seq of rRNA depleted total RNA
(see iLab service: NGS library prep – RNAseq – R)
NGS RNA-seq libraries for the sequencing of rRNA depleted total RNA are prepared using the NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina® with NEBNext® rRNA Depletion Kit (Human/Mouse/Rat). Please provide 100ng–1μg of total RNA in up to 12μl of water for the library prep, plus an additional 4μl for sample QC.
NGS library prep – small RNAseq
Small RNA-seq can be used to sequence many small RNA species such as micro RNA and other small noncoding RNAs. NGS libraries for the sequencing of small RNA are prepared using the NEBNext® Multiplex Small RNA Library Prep kit in conjunction with the Sage Science Pippin Prep for library size selection. Please provide 100 ng–1 μg of total RNA in up to 6 μl of water (160 ng/μl is optimal if possible) for the library prep, plus an additional 4 μl for sample QC.
NGS library prep – 16s Metagenomic
Libraries for 16S Metagenomic Sequencing are prepared using Illumina’s recommended protocol. It allows for multiplexing of up to 96 libraries per sequencing run using Illumina’s dual indexing system. Please provide 5 μl of genomic DNA in 10 mM Tris pH 8.5 at a concentration of 5ng/μl. Please inquire if you would like the core to design a protocol that will allow for a greater number of multiplexing.
Other instrumentation/techniques to assist in either core or user prepared NGS library prep:
Pippin Prep – The Pippin Prep by Sage Science automates gel size selection of libraries or DNA fragments between 100 bp – 1.5 kb. The Pippin Prep is located in Pinn Hall room 1076 and can be run by either the core staff or the customer. The core currently stocks 2% agarose, dye free, external marker cassettes that can accommodate 4 samples. Other cassettes are available from the manufacturer upon request, but will require a minimum number of samples (please inquire). Customers who wish to use the Pippin Prep themselves must be trained and supply all their own supplies and reagents.
Covaris S2 – The Covaris S2 instrument mechanically shears nucleic acids into suitably sized pieces for NGS library prep using focused acoustic energy. This instrument is located in Pinn Hall room 1076 and is user operated. Users must supply all supplies and reagents.