How to Prepare Libraries for Submission:
Labs are responsible for quantifying their own libraries using qPCR (reagents are purchasable from the GenCore) and pooling libraries prior to submission. Researchers must receive an official run confirmation before physical submission of their pool.
- Quantify stock libraries with qPCR and adjust the concentration of the final libraries (according to the table below) using 10 mM Tris-HCL, pH 8.0 + 0.05% Tween 20.
- The Quick Reference Guide for the KAPA Protocol can be found here.
- If quantifying using Qubit, use this calculator to convert ng/uL to nM.
Note: If the library pool concentration is below the submission concentrations listed in the above table, contact GenCore for further instructions and advice.
- To avoid changes in concentration associated with freeze-thaw, libraries should not be frozen before dilution, pooling or submission to GenCore.
- Pools must be submitted in LowBind low retention tubes and labeled with NYU netID, pool name, and concentration.
- Physical submission of the pools must be no more than 5 days prior to the start of the sequencing run. Submission should only occur once the confirmation email has been received.
Custom Sequencing Primers:
- If using custom sequencing primers, these must be submitted along with the samples, and denoted on TuboWeb.
- Aliquots must be at 100uM in at least 20uL and in a LowBind Tube clearly labeled.
Submitting Samples for Library Preparation and Sequencing:
Library preparation requires high quality nucleic acid, for DNA sequencing provide a gel picture of your sample showing High Molecular Weight DNA without degradation, for RNA sequencing provide Bionalayzer trace with RNA Integrity Number (RIN) greater than 8. Concentration must be determined using fluorometric-based method (Quant-IT, Qubit).
- For DNA sequencing, required DNA quantity can be as low as 1 ng and up to 4 µg depending on library type and protocol
- For RNA sequencing usual input is 1 µg of total RNA
- Include a DNase step with the RNA isolation method to ensure purity and accurate quantification of the sample
Resuspend your samples in 10 mM Tris-HCl, pH 8.5 If the minimum amount required can be exceeded, we strongly suggest to increase this amount since a Nanodrop versus Qubit ratio of 10 is frequently observed.
Note: Failing to provide high quality samples in sufficient amount will lead to library preparation failure and/or bad sequencing data. Prior to submitting any sample, please contact the sequencing Core to discuss your project.
Submitting Libraries and Pools for sequencing:
Libraries and Pools must be submitted in low retention tubes, resuspended in 10 mM Tris-HCl, pH 8.5 with 0.1% Tween 20, and with their bioanalyzer trace. If submitting a pool of libraries, please follow the TruSeq Library Prep Pooling Guide for different valid pooling strategies based on plexity. At reception pools will be quantified by qPCR for optimum loading on the Flowcell.
- Custom Sequencing Primers: If using custom sequencing primers, these must be submitted along with samples. Aliquots must be at 100 uM in at least 20 ul Primers must be diluted in EB or DI Water Must be in a Low-bind tube and clearly labeled