cDNA library preparation for nanopore sequencing (SOP ID: 0005)

Revision nr.:
Release date:
November 25, 2019

Last release date:

November 25, 2019
Ada Serena Marletta


Ada Serena Marletta

1. Scope

This Standard Operating Procedure provides a step-by-step protocol for cDNA library preparation for sequencing using MinION (Oxford Nanopore). In detail, steps here described are end-prep of cDNA, barcode ligation and subsequent adapter ligation.

2. Background

This Standard Operating Procedure is intended for qualified personnel who was trained according to the ISO-9001 quality framework.

3. Prerequisites

Documents required:

  • MIAMI quality manual
  • SOP ID 0001

Material required:

  • Generic lab-, glass- and plastic-ware
  • cDNA in 20 μl nuclease-free water
  • NEBNext Ultra II End repair / dA-tailing Module (E7546)
  • 0.2 ml thin-walled PCR tubes
  • Nuclease-free water (e.g. ThermoFisher, cat # AM9937)
  • Freshly prepared 70% ethanol in nuclease-free water
  • 1.5 ml Eppendorf DNA LoBind tubes
  • Agencourt AMPure XP beads
  • Native Barcoding Expansion 1-12 (EXPNBD104) and 13-24 (EXP-NBD114) if multiplexing more than 12 samples
  • NEB Blunt/TA Ligase Master Mix (M0367)
  • Direct cDNA Sequencing Kit (SQK-DCS109)
  • NEBNext Quick Ligation Module (E6056)

4. Changes since last revision

No changes were made since last version

5. Role and responsibilities

Author (Au): Staff scientist properly trained to draft, review and disseminate standard operating procedures within the framework of ISO-9001

Approver (Ap): PI with the authority to approve, reject and withdraw standard operating procedures

MIAMI Quality Manager (QM): MIAMI authorized Quality Manager

Operator (Op): Staff scientist properly trained to execute the standard operating procedure

6. Monitoring Requirements

The present document can be reviewed at any time required by the author and the MIAMI Quality Manager
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