PHCO NextSeq

Introduction

The NextSeq500 was purchased by the Department in August of 2014. It is available for use by all members of the Department. 

For general info on the Illumina platform and the NextSeq, check out the short informational videos on their websiteThere are 3 videos worth watching (even if you don’t plan to use TruSeq kits): Sequencing:Illumina Technology, NextSeq500 Overview, TruSeq:RNA Sample Prep

If you would like to submit samples for sequencing, please look through the information outlined below. Users are responsible for preparing their own samples and purchasing the appropriate reagent kit for the NextSeq.   

Preparing Samples and Quality Control

Purchase a kit through Illumina off of UNC's standing quote, or use another provider, like Kapa (standing quote), or NEB or Agilent. If you are purchasing your own adapters, follow the guidelines listed here to prepare them. If you are curious to learn how adapters work, read this.

Once you have prepared your libraries, quantify the DNA using a fluorometer or nanodrop, and estimate the average size of the library via agarose gel electrophoresis. Then, pool your libraries to a final concentration of 15nM in pH 8/8.5 10mM Tris buffer and submit to Brian for sequencing.

This document outlines some basics of the RNAseq process.

NextSeq Reagents

Illumina currently offers 5 different outputs for the NextSeq(see table below).

To sequence:

NextSeq Kit name:

Max read output:

Illumina Cat #:

single-end 150 OR paired-end 75

NextSeq 500 Mid Output Kit (150 cycles)

1.30E+08

FC-404-2001

single-end 150 OR paired-end 75

NextSeq 500 High Output Kit (150 cycles)

4.00E+08

FC-404-2002

paired-end 150

NextSeq 500 Mid Output Kit (300 cycles)

1.30E+08

FC-404-2003

paired-end 150

NextSeqTM 500 High Output Kit (300 cycles)

4.00E+08

FC-404-2004

single-end 75

NextSeqTM 500 High Output Kit (75 cycles)

4.00E+08

FC-404-2005

Make sure to purchase off of UNC's standing quote. Contact  for a copy.  Before purchasing, let know what you plan to do, and have the reagents sent to:

UNC Dept of Pharmacology
c/o Brian Golitz
4009 Genetic Medicine CB 7365
120 Mason Farm Rd
Chapel Hill NC 27599

Bookings

We have set up a google calendar to sign up for runs. AFTER you have prepared your samples, please book two full days for your run and Brian will contact you about remaining details.

http://www.google.com/calendar
email: nextseq500@gmail.com
password: GMBlab4027