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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