MGISP-960

MGIEasy rRNA Depletion Kit Instructions

Software Version: V1.6.1.301 and later versions

Hardware Version: 2- MGISP-960
Kit Version: V1.2
Automation Version: V1.0

For Research Use Only.

MGI Tech Co., Ltd. All rights reserved
Revision History
Automation Version Date Description

V1.0 June. 2023 Initial release.

NOTE: Please download the latest version of the manual and use it with the corresponding kit.

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Contents
Chapter 1 Product Description......................................................................................................................... 1
1.1 Introduction ................................................................................................................................................... 1
1.2 Software ......................................................................................................................................................... 1
1.3 Hardware ....................................................................................................................................................... 1
1.4 Applicable Reagent Kits ................................................................................................................................ 1
1.5 Equipment and Materials Required but not Provided .................................................................................... 1
1.6 Precautions..................................................................................................................................................... 2
1.7 Principles and Workflow................................................................................................................................ 3
Chapter 2 Applicable Conditions for Automated ........................................................................................... 4
2.1 Sample Compatibility and Requirement ........................................................................................................ 4
2.2 Sample Quality Control ................................................................................................................................. 4
Chapter 3 Standard Workflow of Automated DNA Depletion ...................................................................... 5
3.1 Pre-preparation .............................................................................................................................................. 5
3.2 Instrument Operation ..................................................................................................................................... 7
Chapter 4 Standard Workflow of Automated rRNA Depletion .................................................................... 10
4.1 Pre-preparation ............................................................................................................................................ 10
4.2 Instrument Operation ................................................................................................................................... 12

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Chapter 1 Product Description
1.1 Introduction

MGISP-960 is an automated library preparation system of MGI. This document is intended to guide you to
perform the automated process with the kit (MGIEasy rRNA Depletion Kit V1.2, Cat. No.: 1000005953) on
MGISP-960. The preparation workflow is strictly tested and repeated to ensure maximum stability and
repeatability.

1.2 Software

The applicable software version to this manual is V1.6.1.301.

1.3 Hardware

MGISP-960 customized configuration 2-MGISP-960 platform, including the following hardware:

Table 1- 1 The Hardware of Customized Configuration 2-MGISP-960 Platform

Hardware Quantity& Position
PCR 1 (Pos9~Pos11)
vortex mixer 1 (Pos20)
PCR plate magnetic rack 1 (Pos15)
Deep-well plate magnetic rack 1 (Pos19)
temperature control module 2 (Pos21)
waste container 1 (Pos24)
higher SBS carrier 1 (Pos5)
Lower SBS carrier 1 (Pos7)
Temperature control module (Deep-well plate) 1(Pos21)

1.4 Applicable Reagent Kits

MGIEasy rRNA Depletion kit（32RXN） Cat. No. 1000005953

1.5 Equipment and Materials Required but not Provided

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 Table 1-2 Equipment and Materials Required but not Provided
 Vortex Mixer
 Desktop Centrifuge
Equipment  Plate Centrifuge
 Heat Sealer (Thermo ALPS 50V)
 Pipets
 Milli-Q water
 Nuclease free water (NF water) (Ambion, Cat. No. AM9937)
 100% Ethanol (Analytical Grade)
Reagents
 RNA cleanup beads (Agencourt RNAClean XP 40 mL Kit, AgencourtTM, Cat. No.
A63987)
 DNase I (NEB, Cat. No. M0303S)
 Manual Pipette adapter Tips and RNase-free Tips
Consumables  Non-stick Tube, Non-stick RNase-Free, 1.5 mL Microfuge Tubes（Ambion, Cat.
No. AM12450）

Table 1-3 Customer-prepared Materials for Automation

Consumables Brand Cat. No. Quantity
250 μL automated filter tips MGI 1000000723 14 boxes
1.3 mL U-bottom deep-well plate MGI 1000004644 9 plates
Hard-shell thin-wall 96-well skirted PCR
MGI 1000012059 10 plates
plates, white shell/clear well
Aluminum foil (20 μm Heat Sealing Foil) Vitl V901002 6 sheets

Note： If the customer does not have Heat Sealer (Thermo ALPS 50V), so the aluminum film (20μm
Heat Sealing Foil) not need. When the 96-well plate storing samples or reagents needs to be centrifuged or
temporarily stored, the adhesive transparent sealing film can be used to seal the plate. The adhesive
transparent sealing film needs to be removed before the samples and reagent plates are placed in MGISP-960.

1.6 Precautions

1) Before experiment, read through the operation guide of the related reagent kits.

2) Take out the components from the reagent cartridge prior to use. For enzymes mix, mix well by inversion,
then centrifuge briefly and place on ice until further use. For other buffer mixes, thaw at room temperature,
mix well by vortexing, then centrifuge briefly and place on ice until further use.

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 3) When using the reagents, only the recommended consumables can be used.

4) Before first use, install application scripts according to MGISP-100 & MGISP-960 Application Script
Installation Instructions.

5) Perform a pre-clean after powering on the device and before experiment; perform a post-clean after
experiment and before powering off the device according to MGISP-100 & MGISP-960 Cleaning Instructions.

6) All samples and various wastes should be disposed of in accordance with relevant regulations.

7) If you have other questions, please contact the technical support: MGI-service@mgi-tech.com

1.7 Principles and Workflow

1 h 20 min

RNA Samples

DNA depletion products

DNA depletion reagent

Consumables

Figure 1-1 The Workflow of Automated DNA Depletion

2 h 30 min

RNA Samples

rRNA depletion reagent rRNA depletion products

Consumables

Figure 1-2 The Workflow of Automated rRNA Depletion

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Chapter 2 Applicable Conditions for Automated
2.1 Sample Compatibility and Requirement

This automated library preparation workflow is applicable for total RNA samples from humans and rats. It is
strongly recommended to use 220 ng total RNA for automation.

2.2 Sample Quality Control

1) Use Agilent 2100 Bioanalyzer to perform quality control of extracted total RNA sample. RIN value should be
≥7. If RIN< 7, appropriately increase the RNA input and the number of PCR cycles in the library
preparation.

2) RNA integrity: OD260/OD280=1.8- 2.0, OD260/OD230≥2.

3) If DNA contamination is found in RNA sample, start from the workflow of DNA Depletion.

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Chapter 3 Standard Workflow of Automated DNA Depletion
To ensure correct operation, strictly follow the procedures below:

NOTE: If DNA depletion is included, start reading from chapter 3. If DNA depletion is not included, start
reading from chapter 4.

3.1 Pre-preparation

3.1.1 Preparing the Device

1) Before first use, install the PCR program according to MGISP-100 & MGISP-960 Application Script Installation
Instructions.

2) Perform a pre-clean before experiment according to MGISP-100 & MGISP-960 Cleaning Instructions.

3.1.2 Preparing Consumables

Take out the consumables required for one workflow for further use, as listed in the table below:

Table 3-1 Customer-prepared Materials for Automation

Consumables Brand Cat. No. Quantity
250 μL automated filter tips MGI 1000000723 6 boxes
1.3 mL U-bottom deep-well plate MGI 1000004644 5 plates
Hard-shell thin-wall 96-well skirted PCR
MGI 1000012059 3 plates
plates, white shell/clear well
Aluminum foil (20 μm Heat Sealing Foil) Vitl V901002 3 sheets

3.1.3 Preparing Samples

1) Take out the total RNA samples, thawed on the ice and centrifuge shortly. Dilute the samples to 5.9~6.8
ng/μL is recommended.

2) Take out a new PCR plate (MGI, Cat. No.1000012059), transfer the Normalized RNA sample to the plate,
the final volume is 45 μL, seal the plate with aluminum foil (Vitl, Cat. No. V901002), centrifuge shortly on
plate centrifuge. Ensure that no air bubbles exist at the bottom of the tube and no liquid remains on the
tube wall.

3.1.4 Preparing Reagents

1) Take out the customer-prepared DNase I reagents, prepare 990 μL “DNase I Mix” by using DNase I enzyme
and 10x DNase I Buffer in a 1.5 mL microfuge tube, the volume ratio is 1:2. And thoroughly mix them by using
a vortex mixer and then centrifuge shortly, and place it on ice for further use.

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2) Take out a deep-well plate (MGI, Cat. No. 1000004644), and mark it as “NF Water”. Add reagents according
to the volumes listed in Table 3-2.

Table 3-2 Input Volume of NF Water

Item Consumables Brand Cat. No. Volume/well
NF Water Deep-well plate MGI 1000004644 40 µL

3) 30 min before experiment, take out the customer-prepared RNA cleanup beads, and thoroughly mix them by
using a vortex mixer and then centrifuge shortly.

4) Take out a 96 deep-well plate (MGI, Cat. No.1000004644), and mark it as “DNA Depletion Reagents”. Add
reagents according to the volumes listed in Table 3-3. Seal the plate with aluminum foil (Vitl, Cat. No. V901002)
to prevent condensate from entering the reagent plate. Store the rest of the cleanup beads at 4°C.

Figure 3-1 Deep-well Plate Reagent Distribution

Table 3-3 Actual Distribution Input Amount in Deep-well Plate

Item DNase I Mix RNA Clean Beads RNA Clean Beads / /
Position 1A-1H 2A-2H 3A-3H 4A-4H 5A-5H
Volume 115 μL /well 650 μL /well 650 μL /well / /

5) Prepare 60 mL 80% ethanol by using absolute ethanol and NF water. The 80% ethanol should be used
immediately after preparation.

6) Take out a 96 deep-well plate (MGI, Cat. No. 1000004644), and mark it as “80% Ethanol”. Add reagents
according to the volumes listed in Table 3-4.

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 Table 3-4 Input Volume of UDB PCR Primer Mix
Item Consumables Brand Cat. No. Volume/well
80% Ethanol Deep-well plate MGI 1000004644 500 µL

3.2 Instrument Operation

1) Double-click the icon of MGISP-960 on the desktop. The mode selection interface is displayed, as shown in
the following figure. Select Real and click Login.

Figure 3-2 Mode Selection Interface

2) The initialization interface is displayed.

Figure 3-3 Initialization Interface

3) Click “Initialize”. The initialization takes about 2 minutes. If Initialize successfully is displayed, as shown in the
following figure, the device is connected successfully, and you can go to the next step.

Figure 3-4 Initialization Successful Interface

Note: If the initialization fails, check whether the power switch is turned on, and whether more than
one software program is running. Try to restart the software. If the problem persists, contact technical support.

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4) Click the menu button and select “Run Wizard” in the menu.

5) In the Wizard interface (Figure x-x), click “Solution”, and select “JB-A09-143 MGIEasy FS PCR-Free DNA
Library Prep_RV1.1_SV1.0”, and select “1.MGIEasy_FS_PCRFree_DNA_Library_Prep _step1.py”. Operation
deck arrangement of the first phase is displayed, as shown in following Figure3-6 and Table 3-5. Follow the
on-screen instructions to place the consumables, samples, and reagents, confirm the placement and close
the door.

Figure3-5 Run Wizard Interface

Figure 3-6 Operation Deck Arrangement

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 Table 3-5 First Phase Operation Deck Arrangement
Name Consumables Position
250 μL automated filter tips / Pos 1-Pos6
1.3 mL U-bottom deep-well plate / Pos 18, Pos 23
Hard-shell thin-wall 96-well skirted PCR /
Pos 11，Pos12
plates, white shell/clear well
Hard-shell thin-wall 96-well skirted
RNA sample Pos 14
PCR plates, white shell/clear well
80% Ethanol 1.3 mL U-bottom deep-well plate Pos17
DNA Depletion reagent 1.3 mL U-bottom deep-well plate Pos21
NF-Water 1.3 mL U-bottom deep-well plate Pos22

Note: The sealing film of PCR plates with samples and Library prep reagent need to be removed before
library preparation.

Note: If you use aluminum film (20 μm Heat Sealing Foil) for sealing, please confirm that the
temperature control at Pos21 has a buckle device installed to prevent the deep-well plate from being brought
up when the tip is punctured. If there is no buckle device, remove the sealing film of reagent plate before
operation, and you can also contact MGI after-sales engineer to install the buckle device.

6) Click “Run”, the DNA depletion workflow starts. You can pause or resume the workflow if necessary.

7) After about 1 hour and 20 minutes, take out the DNA depletion products from Pos12，the volume is 20 μL.

8) Dispose of the used deep-well plates, PCR plates, and waste bags to the designated waste area. If no more
experiment is to be conducted on the day, perform a post-clean before powering off the device according to
MGISP-100 & MGISP-960 Cleaning Instructions.

✔ Stopping point: The RNA products plate seal with aluminum foil (Vitl, Cat. No. V901002), stored at
-80 °C for next operation (we recommend that you use the products immediately).

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Chapter 4 Standard Workflow of Automated rRNA Depletion
To ensure correct operation, strictly follow the procedures below:

NOTE: If DNA depletion is included, start reading from chapter 3. If DNA depletion is not included, start
reading from chapter 4.

4.1 Pre-preparation

4.1.1 Preparing Consumables

Take out the consumables required for one workflow for further use, as listed in the table below:

Table 4-1 Customer-prepared Materials for Automation Library

Consumables Brand Cat. No. Quantity
250 μL automated filter tips MGI 1000000723 8 boxes
1.3 mL U-bottom deep-well plate MGI 1000004644 4 plates
Hard-shell thin-wall 96-well skirted PCR
MGI 1000012059 7 plates
plates, white shell/clear well
Aluminum foil (20 μm Heat Sealing Foil) Vitl V901002 3 sheets

4.1.2 Preparing Samples

If DNA depletion is performed, take out the RNA products of step 8 in section 3.5, centrifuge shortly on plate
centrifuge, ensure that no air bubbles exist at the bottom of the tube and no liquid remains on the tube wall,
place it on ice for further use. Otherwise, take out the RNA samples (20 μL,220 ng is recommended) required for
library preparation and pipette them to a PCR plate (MGI, Cat. No. 1000012059), seal the plate with aluminum
foil (Vitl, Cat. No. V901002), centrifuge shortly on plate centrifuge, and then place it on ice for further use. Ensure
that no air bubbles exist at the bottom of the tube and no liquid remains on the tube wall.

4.1.3 Preparing Reagents

1) Take out the following reagents from MGIEasy rRNA Depletion kit, and place them on ice for further use.

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 Table4-2 MGIEasy rRNA Depletion Kit V1.2
Modules &Cat. No. Reagents Spec & Quantity
Probe Mix 68 μL/tube × 1 tube
Hybridization Buffer 227 μL/tube × 1 tube
MGIEasy rRNA Depletion Kit RNase H Buffer 163 μL/tube × 1 tube
(Cat. No. 1000005953) RNase H 91 μL/tube × 1 tube
DNase I Buffer 587 μL/tube × 1 tube
DNase I 187 μL/tube × 1 tube

NOTE: The reagent volumes in Table 4-2 of MGIEasy rRNA Depletion Kit V1.2 is 32 RXN, 3 kits of the same
batch are required to meet the requirements of 96 RXN * 1 time in MGISP-960 automated library
preparation.

2) 30 min before experiment, take out the customer-prepared RNA cleanup beads, and thoroughly mix them
by using a vortex mixer and then centrifuge shortly.

3) Take out a 96 deep-well plate (MGI, Cat. No.1000004644), and mark it as “rRNA Depletion Reagents”. Add
reagents according to the volumes listed in Table 4-3. Seal the plate with aluminum foil (Vitl, Cat. No.
V901002) to prevent condensate from entering the reagent plate. Store the rest of the cleanup beads at 4°C.

Figure 4-1 Deep-well Plate Reagent Distribution

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 Table 4-3 Actual Distribution Input Amount in Deep-well Plate
Hybridization RNase H RNase DNase I
Item DNase I RNA Clean Beads
Buffer Buffer H Buffer
Position 1A-1H 2A-2H 3A-3H 4A-4H 5A-5H 6A-6H
34 μL 220 μL
Volume 85 μL /well 61 μL /well 70 μL /well 500 μL /well
/well /well

RNA Clean
Item / / / / /
Beads
Position 7A-7H 8A-8H 9A-9H 10A-10H 11A-11H 12A-12H
Volume 500 μL /well / / / / /

4) Prepare 60 mL 80% ethanol by using absolute ethanol and NF water. The 80% ethanol should be used
immediately after preparation.

5) Take out two 96 deep-well plates (MGI, Cat. No. 1000004644), and mark them as “80% Ethanol” and “NF
Water” respectively. Add reagents according to the volumes listed in Table 4-4.

Table 4-4 Input Volume of 80% Ethanol and NF Water

Item Consumables Brand Cat. No. Volume/well
80% Ethanol Deep-well plate MGI 1000004644 500 µL
NF Water Deep-well plate MGI 1000004644 40 µL

6) Take out a PCR plate (MGI, Cat. No. 1000012059), and mark it as “Probe Mix”. Add reagents according to the
volumes listed in Table 4-5.

Table 4-5 Input Volume of Probe Mix

Item Consumables Brand Cat. No. Volume/well
Probe Mix PCR plate MGI 1000012059 2 µL

NOTE: Due to the small volume of Probe Mix, please arrange the repacking at the last stage of reagent
preparation.

4.2 Instrument Operation

1) Ensure that MGISP-960 software is started.

2) Click the menu button and select “Run Wizard” in the menu.

3) In the “Run Wizard” interface, click “Solution”, and select “JB-A09-143 MGIEasy RNA Library

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 Prep(250bp)_RV3.1_SV1.0”. Click “Script”, select “2.MGIEasy_rRNA_Depletion_step2.py”. Follow the on-
screen instructions to place the consumables, samples, and reagents, as shown in following figure 4-3 and
table 4-6. Confirm the placement and close the door.

Figure 4-2 Run Wizard Interface

Figure 4-3 Operation Deck Arrangement

Table 4-6 Operation Deck Arrangement

Consumables or Reagents Consumable type Position
250 μL automated filter tips / Pos1-Pos8
Pos12, Pos13,
Hard-shell thin-wall 96-well skirted PCR
/ Pos16, Pos18,
plates, white shell/clear well
Pos23
Hard-shell thin-wall 96-well skirted
Probe Mix Pos11
PCR plates, white shell/clear well
Hard-shell thin-wall 96-well skirted
RNA Sample Pos14
PCR plates, white shell/clear well
1.3 mL U-bottom deep-well plate / Pos19
80% Ethanol 1.3 mL U-bottom deep-well plate Pos17
rRNA Depletion Reagents 1.3 mL U-bottom deep-well plate Pos21
NF Water 1.3 mL U-bottom deep-well plate Pos22

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 Note: The sealing film of PCR plate with samples need to be removed before operation. For reagents plates,
ensure that no bubbles exist at the bottom of the tube and no liquid remains on the tube wall.

Note: If you use aluminum film (20 μm Heat Sealing Foil) for sealing, please confirm that the temperature
control at Pos21 has a buckle device installed to prevent the deep-well plate from being brought up when
the tip is punctured. If there is no buckle device, remove the sealing film of reagent plate before operation,
and you can also contact MGI after-sales engineer to install the buckle device.

4) Click “Run”, the rRNA depletion workflow starts. You can pause or resume the workflow if necessary.

5) After the process runs for about 2 hours and 20 minutes, the RNA elute is processing and new instruction
interface is displayed. And you will be prompted to perform a list of operation, as shown in Figure 4-4.

Figure 4-4 Instructions Interface

6) After the operation completed, confirm that they are correct. Close the instrument door, click “Continue”.
The process of rRNA depletion goes on.

7) After the process runs for about 2 hours and 30 minutes, take out the rRNA depletion products from Pos12，
the volume is 10 μL.

8) Dispose of the used deep-well plates, PCR plates, and waste bag to the designated waste area. If no more
experiment is arranged on that day, perform a post-clean before powering off the device according to
MGISP-100 & MGISP-960 Cleaning Instructions.

✔ Stopping point: The RNA products plate seal with aluminum foil (Vitl, Cat. No. V901002), stored at -80 °C
for automated library preparation (we recommend that you use the products immediately).

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Company: MGI Tech Co., Ltd.
Address: 2/F, Building 11, Beishan Industrial Zone, Yantian District, Shenzhen, CHINA, 518083
Website: http://en.mgi-tech.com
Email: MGI-service@mgi-tech.com
Service Hotline: (+86) 4000-966-988

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