NGS Library Preparation
Library Preparation for bulk sequencing
Core customers can either have the Core prepare their sequencing libraries, typically using a commercially available kit, or can prepare their own libraries using their protocol of choice. The core can work with you to identify or devise a library construction method that fits your 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 library prep – bulk RNAseq – mRNA isolation from total RNA
(see iLab service: NGS library prep – RNAseq – M)
NGS RNA-seq libraries for the sequencing of mRNA are prepared using the NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina® (NEB, #E7760) with the NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB #E7490). Starting material is 10 ng–1 μg of total RNA in a volume of 50 μl. If possible, please provide your samples at the desired final concentration in a 50 μl volume in a 96 well plate. Please provide additional material for QC checking with NanoVue and TapeStation – see the Sample Requirements page for appropriate volumes and concentrations.
NGS library prep – bulk RNAseq – 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™ II Directional RNA Library Prep Kit for Illumina® (NEB, #E7760) and the NEBNext rRNA Depletion Kit (Human/Mouse/Rat) (NEB #E6310). Please provide 10 ng–1 μg of total RNA in up to 12μl of water for the library prep, plus additional material for sample QC with the NanoVue and Tape Station – see the Sample Requirements page for appropriate volumes and concentrations.
NGS library prep – 16s Metagenomic
Libraries for 16S Metagenomic Sequencing are prepared using Illumina’s demonstrated 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 does not currently stock any materials or reagents for use with the Pippin Prep and will not purchase any unless the customer can commit to running a large number of samples. Customers who wish to use the Pippin Prep themselves must be trained and must 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.