Short protocol No.1

Short protocol No.1
SHORT PROTOCOL No. 01 I November 2014
Automated Illumina® TruSeq® Stranded
Total RNA library construction with the
epMotion® 5075t/TMX
Introduction
This protocol describes the configuration and preprogrammed methods for the automated construction of
8/16 or 24 sequencing ready libraries from 100-1000 ng
total RNA with the Illumina TruSeq Stranded Total RNA
kit. The overall hands on time is less than 1 hour, the
total run time of the entire procedure is ~11.5 hours for
24 samples.
The three methods (*.dws files) from the download archive
need to be unzipped prior to importing into the epBlue™
Software. The labware archive (Top Update_TS-strandedTRNA.zip) needs to be imported into the epBlue Software
as well.
The entire library construction procedure is carried out
by 3 sub-methods that need to be run subsequently on
the epMotion. Intermediate products from the individual
sub-methods can be stored at -20 °C for up to 7 days,
according to Illumina’s kit user manual.
Material and Methods
Required equipment
Required consumables
> epMotion 5075 TMX or epMotion 5075t
> additional thermal module (Position C2)
> Gripper
> TS50 pipetting tool
> TS300 pipetting tool
> TM50-8 pipetting tool
> TM300-8 pipetting tool
> 4x thermoadapter for PCR plates, 96-well
> Reservoir rack
> 3x RR Module TC Safe Lock
> PCR Cycler, e.g. Eppendorf Mastercycler ® Pro S
> Alpaqua® LE magnet plate (low elution volume magnet,
Alpaqua order no. A000350)
➡ this magnet is the only one known to work with the low
elution volumes required in some steps of the procedure don’t use an other one!
> epT.I.P.S.® Motion 50 μL Filter
> epT.I.P.S. Motion 300 μL Filter
> Eppendorf twin.tec® PCR plates, 96-well, semi skirted
> Eppendorf twin.tec PCR plates, 96-well, skirted
(for the Index Adapters)
> Eppendorf Safe-Lock Tube 1.5 mL
> Eppendorf Safe-Lock Tube 2.0 mL
> epMotion reservoir 30 mL
> Eppendorf 400 mL reservoir
> Agencourt ® AMPure® XP beads
(Beckman Coulter ®, order nos. A63880, A63881, A63882)
> Agencourt RNAclean® XP beads
(Beckman Coulter, order nos. A63987, A66514)
> 70 and 80 % Ethanol
> RNase free water
> mineral oil, PCR/molecular biology grade
(Sigma-Aldrich®, order no. M5904-500ML)
> SuperScript ® II Reverse Transcriptase
(Life Technologies®, order no. 18064-014)
> Illumina TruSeq Stranded Total RNA kit
(either Ribo-Zero™ or Ribo-Zero Gold)
SHORT PROTOCOL I No. 01 I Page 2
Method
Method Name
approx. Runtime
(24 samples)
XXYYZZ-01-TS-STRNA.dws
5.5hrs
XXYYZZ-02-TS-STRNA.dws
4hrs
XXYYZZ-03-TS-STRNA.dws
2hrs,
including external PCR
Important: The output plate containing the samples that
need to be processed in the subsequent sub-method will
always be placed on the C2 position (Temp) set to 10 °C
at the end of the individual methods. Final libraries will
be found in columns 10-12 of the plate labeled PCR. The
volume is 17.5 μL:
(XX = year, YY = month, ZZ = day)
This approach is programmed to provide as much automation
as possible, thus only up to 24 samples can be processed.
Please only process 8/16 or 24 samples, sample numbers
non divisible by 8 are not supported. The entire workflow
– from 100 to 1000 ng total RNA sample input to sequencing
ready libraries – is divided into three epMotion methods
(or sub-methods, see above). Each of the methods ends at
a “Safe Stopping Point”, allowing to store the intermediate
products at -20 °C to -15 °C for up to 7 days, as stated in the
Kit’s user guide. In the default setup only the third sub-method
requires a user intervention to perform the enrichment PCR
in an external cycler. To avoid dead volumes, all Illumina Kit
reagents are programmed either in 1.5 mL or 2 mL Tubes,
only Agencourt Magnetic Beads and Ethanol for the washes
are provided in 30 mL reservoirs to allow 8 channel pipetting.
All liquid waste is collected in a 400 mL reservoir in B0. As
most of the used volumes are very low, all reagents must
be checked for foam, air bubbles etc. to ensure best performance prior to starting the runs. For some of the reagents,
the beads and the mineral oil, it is mandatory to let them
reach ambient temperature to ensure proper function and
pipetting due to changes in viscosity. During the procedure
no cooling of the reagents is required.
All steps of the procedure are performed in 96-well twin.tec
PCR plates (semi-skirted), for the multiple heat incubation
steps above 37 °C, samples are overlaid with oil to avoid
evaporation and allow temperature incubations on the
epMotion. Only the enrichment PCR needs to be carried
out in an external PCR cycler.
The methods were developed on the epMotion 5075 TMX,
but can also be transferred to the epMotion 5075t.
Figure 1: Position of the final libraries in the plate labeled PCR after
completion of the entire procedure.
SHORT PROTOCOL I No. 01 I Page 3
Sub-method 01
Start with 100 – 1000 ng total RNA in a volume of 10 μL per sample. 8/16 or 24 samples have to be provided in the first
three columns (Wells A1-H3) of a 96-well twin.tec semi skirted PCR plate (BRP+DFP), placed on the TMX position. The
method ends with clean cDNA in the first three columns (A1-H3) of a second 96-well twin.tec semi skirted PCR plate (ALP).
This plate needs to be used in the sub-method 02.
Worktable Layout
Position
Item
Position
Item
A2
50 μL Filtertips
B3
50 μL Filtertips
A3
50 μL Filtertips
B4
Thermoadapter PCR 96
A4 (TMX)
Thermoadapter PCR 96 + PCR plate
with RNA samples (labeled BRP+DFP)
C1
Thermoadapter PCR 96 + empty PCR
plate (labeled ALP)
B0
400 mL tub for liquid waste
C2 (Temp)
Thermoadapter PCR 96
B1
300 μL Filtertips
C3
B2
300 μL Filtertips
Reservoir rack with 3x RR Module Safe
Lock + 4x 30 mL reservoir for reagents
C4
Alpaqua LE magnet plate + empty PCR
plate (labeled RRP-RCP)
Figure 2: epMotion worktable layout for method 1
SHORT PROTOCOL I No. 01 I Page 4
Reservoir rack layout
Resusp.
Buffer
rRNA
removal
Mix
First
Strand
Synth.
Mix*
Mineral Oil
rRNA
removal
Beads
Second
Strand
marking
Mix
Elution
Buffer
End
Repair
Control**
80 % Ethanol, 30mL Reservoir
Elute,
Prime,
Frag.
High Mix
70 % Ethanol, 30 mL Reservoir
rRNA
Binding
Buffer
AMPure XP Beads, 30 mL Reservoir
2 mL SafeLock
RNAClean XP beads, 30 mL Reservoir
1.5 mL SafeLock
Reservoir rack part 1
* with SuperScript RT (1 μL RT + 9 μL FSA Mix)
** 1:50 (not 100!) diluted in Resuspension Buffer
Figure 3: Reservoir rack layout for method 1
Attention: If kit included controls are not going to be used, use plain resuspension buffer in the according reagent position.
SHORT PROTOCOL I No. 01 I Page 5
Sub-method 02
Start with the PCR plate labeled ALP containing the cDNAs from sub-method 01 in Positions A1 – H3, placed on position
B4. The method ends with A-tailed and Index Adapter ligated clean cDNA in a PCR plate labeled PCR. Depending on the
sample number, sequencing setup, pooling scheme etc. the number, combination and labware of the Index Adapters
(position B3) needs to be modified ➡ also review/adjust steps 26 and following + the worktable in the method. If the
default setup is being used, a user intervention to refill 50 μL tips is required.
Worktable Layout
Position
Item
Position
Item
A2
50 μL Filtertips
C1
A3
50 μL Filtertips
Thermoadapter PCR 96 +
empty PCR plate (labeled PCR)
A4 (TMX)
Thermoadapter PCR 96
C2 (Temp)
Thermoadapter PCR 96
B0
400 mL tub for liquid waste
C3
B1
300 μL Filtertips
Reservoir rack with 3x RR Module
SafeLock + 2x 30 mL Reservoir
(pos.5 & 7)
B2
300 μL Filtertips
C4
Alpaqua LE magnet plate
B3
skirted PCR plate with index adapters
➡ review method programming
B4
Thermoadapter PCR 96 + PCR plate with
cDNA (ALP) from sub-method 01
Figure 4: epMotion worktable layout for method 2
SHORT PROTOCOL I No. 01 I Page 6
Reservoir rack layout
Resusp.
Buffer
Ligation
Mix
Ligation
Control **
Mineral Oil
Stop
Ligation
Buffer
80 % Ethanol, 30 mL Reservoir
A-Tailing
Control **
Leave slot empty
A-Tailing
Mix
AMPure XP Beads, 30 mL Reservoir
2 mL SafeLock
Leave slot empty
1.5 mL SafeLock
Reservoir rack part 2
** 1:100 diluted in Resuspension Buffer
Figure 5: Reservoir rack layout for method 2
Attention: If kit included controls are not going to be used, use plain resuspension buffer in the according reagent position.
SHORT PROTOCOL I No. 01 I Page 7
Sub-method 03
Start with 20 μL A-tailed and Index Adapter ligated samples in plate labeled PCR from sub-method 02 on TMX position.
Only the PCR setup will be pipetted, which takes a couple of minutes, then a user intervention occurs where the PCR plate
needs to be sealed and cycled in a PCR cycler to enrich the libraries. Reopen the plate after PCR and restore on the TMX
position prior to continuing the method.
Worktable Layout
Position
Item
Position
Item
A2
50 μL Filtertips
C1
Thermoadapter PCR 96
A3
empty
C2 (Temp)
Thermoadapter PCR 96
A4 (TMX)
Thermoadapter PCR 96 + PCR plate
(PCR) with samples from sub-method 02
C3
B0
400 mL tub for liquid waste
Reservoir rack with 3x RR Module
Safe Lock + 2x 30 mL Reservoir
(pos.5 & 7)
B1
300 μL Filtertips
C4
Alpaqua LE magnet plate
B2
300 μL Filtertips
B3
empty
B4
Thermoadapter PCR 96
Figure 6: epMotion worktable layout for method 3
SHORT PROTOCOL I No. 01 I Page 8
Reservoir rack layout
Leave slot empty
PCR
Master
Mix
80 % Ethanol, 30 mL Reservoir
Resusp.
Buffer
Leave slot empty
Primer
Cocktail
2 mL SafeLock
AMPure XP Beads, 30 mL Reservoir
1.5 mL SafeLock
Reservoir rack part 3
Figure 7: Reservoir rack layout for method 3
After all three sub-methods have been completed, the final libraries are in the plate labeled PCR on position C2 (Temp)
at 10 °C.
SHORT PROTOCOL I No. 01 I Page 9
Ordering Information
Description
Order no. international
epMotion 5075t
5075 000.302
Thermal module
5075 757.001
TS 50 dispensing tool
5280 000.010
TS 300 dispensing tool
5280 000.037
TM50-8 dispensing tool
5280 000.215
TM300-8 dispensing tool
5280 000.231
Gripper
5282 000.018
Thermoadapter PCR 96
5075 787.008
Reservoir rack
5075 754.002
Reservoir rack module TC Safe-Lock
5075 799.081
epT.I.P.S.® Motion, 50 µL, filtered
0030 014.413
epT.I.P.S. Motion, 300 µL, filtered
0030 014.456
Reservoir 30 mL
0030 126.505
Reservoir 400 mL
5075 751.364
®
®
Eppendorf twin.tec® PCR Plate 96, semi-skirted
0030 128.575
Eppendorf twin.tec PCR Plate 96, skirted
0030 128.648
Eppendorf Safe-Lock Tubes, 1.5 mL
0030 120.086
Eppendorf Safe-Lock Tubes, 2.0 mL
0030 120.094
®
Your local distributor: www.eppendorf.com/contact
Eppendorf AG · 22331 Hamburg · Germany
[email protected] · www.eppendorf.com
www.eppendorf.com/automation
Alpaqua® is a registered trademark of Alpaqua Engineering, LLC., USA. Illumina® and TruSeq® are registered trademarks of Illumina, Inc., USA. Ribo-Zero™ is a trademark of Illumina, Inc., USA. Agencourt®, AMPure®,
Beckman Coulter® and RNAclean® are registered trademarks of Beckman Coulter, Inc., USA. Sigma-Aldrich® is a registered trademark of Sigma-Aldrich Co., LLC., USA. Superscript® and Life Technologies® are registered
trademarks of Life Technologies Corporation, USA. Eppendorf®, the Eppendorf logo, epMotion®, epT.I.P.S.®, Mastercycler® and Eppendorf twin.tec® are registered trademarks of Eppendorf AG, Germany.
epBlue™ is a trademark of Eppendorf AG, Germany. U.S. Design Patents are listed on www.eppendorf.com/ip. All rights reserved, including graphics and images. Copyright © 2014 by Eppendorf AG, Hamburg, Germany.
Methods are intended for molecular research applications. They are not intended, verified or validated, for use in the diagnosis of disease or other human health conditions.
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