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

SOP ID:

0005

Revision nr.:

0.0

Release date:

November 25, 2019

Last release date:

November 25, 2019

Author(s):

Ada Serena Marletta

Approver:

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

7. Procedure

Step	Action	Responsibility
1	Mix the following reagents in a sterile nuclease-free 0.2 ml PCR tube: 20 µl cDNA sample prepared following PRCD 0003 30 µl Nuclease-free water 7 µl Ultra II End-prep reaction buffer 3 µl Ultra II End-prep enzyme mix 60 µl Total	Op
2	Set a 100 µl or 200 µl pipette to 50 µl and then pipette the entire volume up and down at least 10 times to mix thoroughly. Perform a quick spin to collect all liquid from the sides of the tube. The presence of a small amount of bubbles will not interfere with performance.	Op
3	Using a thermal cycler, incubate at 20° C for 5 minutes and 65° C for 5 mins.	Op
4	Prepare the AMPure XP beads for use; resuspend by vortexing.	Op
5	Transfer the sample to a 1.5 ml DNA LoBind Eppendorf tube.	Op
6	Add 60 µl of resuspended AMPure XP beads to the end-prep reaction and mix by pipetting.	Op
7	Incubate on a Hula mixer (rotator mixer) for 5 minutes at RT	Op
8	Prepare 500 μl of fresh 70% ethanol in Nuclease-free water.	Op
9	Spin down the sample and pellet on a magnet. Keep the tube on the magnet, and pipette off the supernatant.	Op
10	Keep on magnet, wash beads with 200 µl of freshly prepared 70% ethanol without disturbing the pellet. Remove the 70% ethanol using a pipette and discard.	Op
11	Repeat step 10.	Op
12	Spin down and place the tube back on the magnet. Pipette off any residual ethanol. Allow to dry for ~30 seconds, but do not dry the pellet to the point of cracking.	Op
13	Remove the tube from the magnetic rack and resuspend pellet in 22.5 µI Nuclease-free water. Incubate for 2 minutes at RT.	Op
14	Pellet the beads on a magnet untiI the eluate is clear and colourless.	Op
15	Remove and retain 22.5 µI of eluate into a clean 1.5 ml Eppendorf DNA LoBind tube.	Op
16	Take forward 22.5 µI of end-prepped cDNA into barcode ligation.	Op
17	Add the reagents in the order given below, mixing by flicking the tube between each sequential addition: 22.5 µI End-prepped DNA 2.5 µI Native Barcode 25 µI Blunt/TA Ligase Master Mix 50 ul Total	Op
18	Mix gently by flicking the tube, and spin down.	Op
19	Incubate the reaction for 10 minutes at RT.	Op
20	Prepare the AMPure XP beads for use; resuspend by vortexing	Op
21	Add 50 µI of resuspended AMPure XP beads to the end-prep reaction and mix by pipetting.	Op
22	Incubate on a Hula mixer (rotator mixer) for 5 minutes at RT.	Op
23	Prepare 500 µI of fresh 70% ethanol in Nuclease-free water.	Op
24	Spin down the sample and pellet on a magnet. Keep the tube on the magnet, and pipette off the supernatant.	Op
25	Keep on magnet, wash beads with 200 µI of freshly prepared 70% ethanol without disturbing the pellet. Remove the 70% ethanol using a pipette and discard.	Op
26	Repeat step 25.	Op
27	Spin down and place the tube back on the magnet. Pipette off any residual 70% ethanol. Briefly allow to dry.	Op
28	Remove the tube from the magnetic rack and resuspend pellet in 26 µI Nuclease-free water. Incubate for 2 minutes at RT.	Op
29	Pellet beads on magnet until the eluate is clear and colourless	Op
30	Remove and retain 26 µI of eluate into a clean 1.5 ml Eppendorf DNA LoBind tube.	Op
31	Quantify 1 µI of eluted sample using a Qubit fluorometer.	Op
32	Pool the barcoded samples at the desired ratio to a final volume of 65 μl in a DNA LoBind 1.5ml Eppendorf tube. Aim for as high a concentration as possible which does not exceed 200 fmoles total. If the total volume is >65 μl, perform a 2.5x AMPure clean up and elute in 65 μl of nuclease-free water.	Op
33	Thaw Adapter Bead Binding (ABB) Buffer, Elution Buffer (EB) and NEBNext Quick Ligation Reaction Buffer (5x) at room temperature, mix by vortexing, spin down and place on ice. Check the contents of each tube are clear of any precipitate.	Op
34	Spin down the T4 Ligase and the Adapter Mix II (AMII), and place on ice.	Op
35	Taking the pooled and barcoded DNA, perform adapter ligation as follows, mixing by flicking the tube between each sequential addition: 65 µI 200 fmol pooled barcoded sample 5 µl Adapter Mix II (AMII) 20 µI NEBNext Quick Ligation Reaction Buffer (5X) 10 µI Quick T4 DNA Ligase 100 µl Total
36	Mix gently by flicking the tube, and spin down.	Op
37	Incubate the reaction for 10 minutes at RT.	Op
38	Prepare the AMPure XP beads for use; resuspend by vortexing.	Op
39	Add 50 µl of resuspended AMPure XP beads to the reaction and mix by pipetting.	Op
40	Incubate on a Hula mixer (rotator mixer) for 5 minutes at room temperature	Op
41	Place on magnetic rack, allow beads to pellet and pipette off supernatant.	Op
42	Add 140 µI of the Adapter Bead Binding (ABB) buffer to the beads. Close the tube lid, and resuspend the beads by flicking the tube. Return the tube to the magnetic rack, allow beads to pellet and pipette off the supernatant.	Op
43	Repeat step 42.	Op
44	Remove the tube from the magnetic rack and resuspend pellet in 13 µI Elution Buffer (EB).	Op
45	Incubate on a Hula mixer (rotator mixer) for 10 minutes at room temperature.	Op
46	Pellet beads on magnet until the eluate is clear and colourless	Op
47	Remove and retain 13 µl of eluate into a clean 1.5 ml Eppendorf DNA LoBind tube.	Op
48	Quantify 1 µl of eluted sample using a Qubit fluorometer	Op
49	The prepared library is used for loading into the MinlON flow cell. Store the library on ice until ready to load.	Op

8. Record Management

When completed and approved, the SOP will be published on MIAMI website

9. References

9.1 Standards

ISO-9001:2015
International System of Units

9.2 Other SOPs

The execution of this SOP refer to cDNA preparation for nanopore sequencing (SOP ID: 0003)

9.3 Reagents

For reference reagents, please refer to the Prerequisites section.

9.4 Sequences

The execution of this SOP does not refer to any reference sequence

9.5 Supplementary Documents

This SOP is an adaptation of Direct cDNA Native Barcoding Protocol available on Nanopore’s website.

10. Definitions

Measurements units are defined according to International System of Units

Metric prefixes are defined according to Metric (SI) Prefixes

Whenever possible, biological construct are defined according to the SBOL standard

Whenever possible, biological experiments are defined according to the metrology standards

11. Disclaimers

This Standard Operating Procedure (SOP) was developed based on a compilation of best available information, knowledge, experience, and general industry practices to provide guidance to MIAMI staff in performing the activities defined herein, in a consistent and standardized manner. This document does not contain regulatory or statutory requirements unless specified. The MIAMI consortium has made every attempt to present the information in a clear and concise manner for a variety of users. However, the MIAMI consortium is not responsible for the misuse or misinterpretation of the information presented herein. Under no circumstances shall The MIAMI consortium be liable for any actions taken or omissions made by non-MIAMI users of this document. In general, this document should be used as a reference. Differences may exist between the procedures referenced in this document and what is appropriate under project-specific conditions. This document does not represent an endorsement of practitioners or products mentioned in the document.

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