cDNA preparation for nanopore sequencing (SOP ID: 0003)

SOP ID:

0003

Revision nr.:

0.0

Release date:

November 19, 2019

Last release date:

November 29, 2019

Author(s):

Ada Serena Marletta

Approver:

Ada Serena Marletta

1. Scope

This Standard Operating Procedure provides a step-by-step protocol for cDNA synthesis starting from polyA mRNA. The synthesized cDNA is then subjected to Oxford Nanopore sequencing.

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
100 ng PolyA+ RNA
Direct cDNA Sequencing Kit (SQK-DCS109)
Agencourt AMPure XP beads
1.5 ml Eppendorf DNA LoBind tubes
0.2 ml thin-walled PCR tubes
Nuclease-free water (e.g. ThermoFisher, cat #AM9937)
Freshly prepared 70% ethanol in nuclease-free water
10 mM dNTP solution (e.g. NEB N0447)
LongAmp Taq 2X Master Mix (e.g. NEB M0287)
Maxima H Minus Reverse Transcriptase (200 U/µl) with 5x RT Buffer (ThermoFisher, cat # EP0751)
RNaseOUT™, 40 U/μl (Life Technologies, 10777019)
RiboShredder (Epicentre, RS12500), or RNase Cocktail Enzyme Mix (ThermoFisher, AM2286)

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	Transfer 100 ng PolyA+ RNA into a DNA LoBind tube	Op
2	Adjust the volume to up to 7.5 μl with Nuclease-free water	Op
3	Mix by flicking the tube to avoid unwanted shearing and spin down briefly in a microfuge	Op
4	In a 0.2 ml PCR tube, mix the following: 2.5 μl VN Primer (VNP) 1 μl 10 mM dNTPs 7.5 μl PolyA+ RNA prepared in Nuclease-free water	Op
5	Mix gently by flicking the tube, and spin down.	Op
6	Incubate at 65° C for 5 minutes and then snap cool on a pre-chilled freezer block.	Op
7	In a separate tube, prepare the strand-switching buffer by mixing the following: 4 μl 5x RT Buffer 1 μl RNaseOUT 1 μl Nuclease-free water 2 μl Strand-Switching Primer (SSP)	Op
8	Mix gently by flicking the tube, and spin down.	Op
9	Add the strand-switching buffer prepared at step 7 to the snap-cooled, annealed mRNA, incubated at step 6	Op
10	Mix by flicking the tube and spin down briefly in a microfuge	Op
11	Incubate at 42° C for 2 minutes.	Op
12	Add 1 µl of Maxima H Minus Reverse Transcriptase. The total volume is now 20 µl.	Op
13	Mix gently by flicking the tube, and spin down briefly in a microfuge	Op
14	Incubate the reverse transcription reaction as follows: Reverse transcription and strand-switching 90 mins @ 42° C (1 cycle) Heat inactivation 5 mins @ 85° C (1 cycle) Hold @ 4° C	Op
15	Add 1 µl RiboShredder or RNase Cocktail Enzyme Mix (ThermoFisher, AM2286) to the reverse transcription reaction	Op
16	Incubate the reaction for 10 minutes at 37° C.	Op
17	Prepare the AMPure XP beads for use; resuspend by vortexing.	Op
18	Transfer the sample to a clean 1.5 ml Eppendorf DNA LoBind tube.	Op
19	Add 17 µl of resuspended AMPure XP beads to the reaction and mix by flicking the tube.	Op
20	Incubate on a Hula mixer (rotator mixer) for 5 minutes at RT.	Op
21	Prepare 500 μl of fresh 70% ethanol in Nuclease-free water.	Op
22	Spin down the sample and pellet on a magnet. Keep the tube on the magnet, and pipette off the supernatant.	Op
23	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
24	Repeat step 23.	Op
25	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
26	Remove the tube from the magnetic rack and resuspend pellet in 20 µl Nuclease-free water.	Op
27	Incubate on a Hula mixer (rotator mixer) for 10 minutes at RT.	Op
28	Pellet beads on magnet until the eluate is clear and colourless	Op
29	Remove and retain 20 µl of eluate into a clean 1.5 ml Eppendorf DNA LoBind tube.	Op
30	Prepare the following reaction in a 0.2 ml thin-walled PCR tube: 25 μl 2x LongAmp Taq Master Mix 2 μl PR2 Primer (PR2) 20 μl Reverse-transcribed sample from above 3 μl Nuclease-free water	Op
31	Incubate the reaction as follows: 94 °C 1 min 50 °C 1 min 65 °C 15 mins 4 °C ∞	Op
32	Prepare the AMPure XP beads for use; resuspend by vortexing.	Op
33	Transfer the sample to a clean 1.5 ml Eppendorf DNA LoBind tube.	Op
34	Add 40 µl of resuspended AMPure XP beads to the reaction and mix by flicking the tube.	Op
35	Repeat steps 20-25	Op
36	Remove the tube from the magnetic rack and resuspend pellet in 21 µl Nuclease-free water.	Op
37	Incubate on a Hula mixer (rotator mixer) for 10 minutes at RT.	Op
38	Pellet beads on magnet until the eluate is clear and colourless.	Op
39	Remove and retain 21 µl of eluate into a clean 1.5 ml Eppendorf DNA LoBind tube.	Op
40	Analyse 1 µl of the strand-switched DNA for size, quantity and quality.	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 does not refer to any other SOP

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 an example protocol using Direct cDNA sequencing kit 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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