IFU-AK0025-8 Archer Universal RNA Reagent Kit Ion
Contents
1. Zi X W K t LN Instructions for Use ARCHER by enzymatics Archer Universal RNA Fusion Detection for lon lorrent Platform AK0025 8 Table of Contents Archer Universal RNA Reagent Kit for lon Torrent Platform 1 TEO Be aha oc eee eee nn nee nn E ee ee ere eee eee eee ee eee eee 1 PO EDE CAPTION a E E Pe te eee et een eon eee ee 2 MOGUA T rea eO BR ee ee ne a OE 7 Wore On OVET IC W ee E EER EN E 2 Version Additions and Changes scssiressirasiaini sarian ee dpa eque EERE 3 K CORO e e EE E E OEA E E O A E E E E E 3 Materials Required But Not Supplied oneris ctn erepta tmu aeaee aAA EAE EAE EE RBRB 3 Conor EOIN Sc T T T E E E EE E E eM EE E 3 SE e A A AE EAT A NA EAA E DEA E 2o 4 Sample gg sosesc E EE E E E EEEE EE E TAER EEEE EEEE 4 Input Nucleic Acid Concentration and PUNTO DET cores sescho acte ibant hte better tatto reto eite trae eaae be obar rcu tb terra nncs 4 ssim cmo UNDIS cf ENDE REN E E Pee CP On ge E E Bee gee E eee ee D M HUG on a e E E E E E ES 0 Oe EUH E 5 Step 2 First Strand cDNA Synthesis sc csusccessepuaransatosiennicsusadetbennsiitonateronnddiaratotdennassny ss trementes Rosa 5 olp oecon otan CONA S TOSS ence ee EER D E MI DEM MEE a UU SUE 6 SrA NA VEI T ea E E E E E 7 Weide Oe AT L a O2. 20 uL Transfer _ 24 uL Transfer Quantitate Library and Sequence 2 Archer Universal RNA Reagent Kit for lon Torrent Platform IFU AK0025 8 Rev B ARCHER gt by enzymatics Version Additions amp Changes e Added multiplexing recommendations for the Archer FusionPlex Heme and Sarcoma Panels Kit Contents 500 mM Tris HCl pH 8 0 SA0020 Ultra Pure Water SA0021 Ultra Pure Water for Ethanol Dilution SA0022 Lyophilized Reagents a Step 1 Random Priming SA0001 Step 2 First Strand cDNA Synthesis SA0002 Step 3 Second Strand cDNA Synthesis SA0003 Step 4 End repair dA tailing SA0004 Step 5 Adapter Ligation SA0005 Step 6 First PCR SA0011 Step 7 Second PCR SA0015 pe QM TS pe go oolosc Materials Required But Not Supplied 1 Archer MBC Adapters for lon Torrent Archer FusionPlex Assay Cat AK0028 8 AK0028 9 AK0032 8 Agencourt AMPure XP Beads Cat A63881 Life Technologies DynaMag Cat 12331D 10096 ethanol ACS grade KAPA Biosystems Library Quantification Kit lon Torrent Universal Cat KK4827 Custom Primer Panels designed at http assay enzymatics com If nucleic acid is from FFPE tissue it is recommended to use Agencourt FormaPure A33342 for extraction Go C S amp S ot E c9 iu General Precautions e Read the entire protocol before beginning e Take note of stopping points where samples can be frozen at 20 C and plan your
3. 8 O E T e EA EEA O AE EEEE A AE EA ER EA E A E g SII AET MITES RE 10 Step 8 Quantify Library and Sequence 12 For more information please visit http www enzymatics com archer ssssssssssn 13 1 Archer Universal RNA Reagent Kit for lon Torrent Platform IFU AK0025 8 Rev B ARCHER by enzymatics Product Description Gene fusions represent an important class of genomic rearrangements in translational research The Archer Universal RNA Reagent Kits and FusionPlex assays utilize the power of next generation sequencing to improve the detection of genomic rearrangements over traditional methods such as immunohistochemistry IHC and fluorescence in situ hybridization FISH Modular Assay Format The Universal RNA Reagent Kit used in conjunction with Archer Assays and MBC Adapters allows users to construct lon Torrent sequencing platform ready libraries from total nucleic acid or RNA samples Universal RNA Reagent Kit FusionPlex Assays MBC Adapters E For Research Use Only Not for use in diagnostic procedures Workflow Overview 20 uL of TNA RNA H O Random Priming WM Www vow wo 20 uL Transfer First Strand cDNA Synthesis 20 uL Transfer 20 uL H20 40 uL Transfer End Repair dA Tailing 40 uL Transfer 10 uL H20 nw MBC Adapters Adapter Ligation 50 uL Transfer 20 uL Transfer First PCR RAPER 20 uL Transfer Purification aa 20 uL Transfer
4. Collect beads with magnet for 2 4 minutes or until solution is clear 5 11 Carefully pipette off and discard supernatant without disturbing the beads 5 12 Wash twice with 200 uL of 70 ethanol while on the magnet Spin down and carefully remove remaining supernatant while taking care to not resuspend beads 8 Archer Universal RNA Reagent Kit for lon Torrent Platform IFU AK0025 8 Rev B ARCHER gt by enzymatics 5 13 After the second wash dry beads at room temperature for 5 minutes 5 14 Elute cDNA in 24 uL of 10 mM Tris HCl Remove tubes from the magnet and thoroughly resuspend the beads with the 10 mM Tris HCl 5 15 Place cDNA bead solution back on magnet for 2 minutes 5 16 Carefully transfer 22 uL of the purified library solution to a fresh 200 uL PCR tube or proceed directly to otep 6 Be sure to avoid transferring beads to the fresh tube Stopping point It is OK to stop and store the library at 20 C Step 6 First PCR NOTE The Archer Universal RNA Reagent Kits do not contain gene specific primers GSPs in the reaction pellet 6 1 Gently open the First PCR 840011 foil pouch by tearing along the indents located at the top of the silver package 6 2 Remove the clear 8 tube strip Each tube in the strip provides a single reaction 6 3 Centrifuge briefly to ensure lyophilized material is in the bottom of the tube 6 4 If you would like to use fewer than eight reactions detach the appropr
5. reactions detach the appropriate number of tubes carefully using clean scissors or a new razor blade Store the remaining unused tubes in the sealed foil pouch with desiccant provided at 4 C Place the tubes on ice and to each add Ultra Pure Water SA0021 20 X uL Purified Total Nucleic Acid X uL Total 20 uL After the lyophilized pellet dissolves gently pipet up and down 6 8 times and briefly spin down Transfer the tubes from ice to the thermal cycler and incubate at 65 C for 5 minutes Remove tubes from thermal cycler and place on ice for 2 minutes then briefly centrifuge before proceeding with First Strand DNA Synthesis otep 2 First Strand cDNA Synthesis 2 1 2 2 Gently open the First Strand cDNA Synthesis SA0002 foil pouch by tearing along the indents located at the top of the silver package Remove the purple 8 tube strip Each tube in the strip provides a single reaction b Archer Universal RNA Reagent Kit for lon Torrent Platform IFU AK0025 8 Rev B ARCHER gt by enzymatics 2 3 Centrifuge briefly to ensure lyophilized material is in the bottom of the tube 2 4 f you would like to use fewer than eight reactions detach the appropriate number of tubes carefully using clean scissors or a new razor blade Store the remaining unused tubes in the sealed foil pouch with the desiccant provided at 4 C 2 5 Place the First Strand cDNA Synthesis tubes on ice and transfer 20 uL of the Rand
6. workflow accordingly e Use good laboratory practices to minimize cross contamination of nucleic acid products e Always use PCR tubes microfuge tubes and pipette tips that are certified sterile DNase and RNase free e Before starting wipe down work area and pipettes with an RNase and DNA cleaning product such as RNase Away Molecular BioProducts Inc San Diego CA e For consistent library amplification ensure the thermal cycler used in this protocol is in good working order and has been calibrated to within the manufacturer s specifications 3 Archer Universal RNA Reagent Kit for lon Torrent Platform IFU AK0025 8 Rev B ARCHER gt by enzymatics Storage All components of Archer Universal RNA Reagent Kit Part AK0025 8 should be stored at 4 C Allow pouches to warm to room temperature before opening sample Multiplexing In order to efficiently utilize the throughput of the PGM multiple samples should be sequenced simultaneously Samples can be identified through a unique nucleotide sequence that is part of the adapter attached to the nucleic acid molecule in a given sample during library construction and which is subsequently read during the sequencing process The unique nucleotide sequence is often termed an index Archer Universal RNA Reagent Kit for lon Torrent Platform utilizes a single index to distinguish between samples The index is added just before Step 5 Adapter Ligation and i
7. Analysis Pipeline http archer enzymatics com 12 Archer Universal RNA Reagent Kit for lon Torrent Platform IFU AK0025 8 Rev B ARCHER by enzymatics Limitations of Use For Research Use Only Not for use in diagnostic procedures his product was developed manufactured and sold for in vitro use only The product is not suitable for administration to humans or animals SDS sheets relevant to this product are available upon request 2014 Enzymatics Inc All rights reserved Archer and Archer FusionPlex are trademarks of Enzymatics Inc lon Torrent PGM Life Technologies and DynaMag are registered trademarks of Thermo Fisher scientific Inc Agencourt AMPure and FormaPure are registered trademarks of Agencourt Biosciences Corporation a Beckman Coulter company KAPA Biosystems is a registered trademark of KAPA Biosystems Inc RNase Away is a registered trademark of Molecular Bio Products Inc For more information please visit http www enzymatics com archer a Enzymatics Inc 2 9 t 100 Cummings Center Suite 407J cl yl NaWC S Beverly MA 01915 e Phone 888 927 7027 13 Archer Universal RNA Reagent Kit for lon Torrent Platform IFU AK0025 8 Rev B
8. above 16 C Stopping point It is OK to stop and store the library at 20 C 6 Archer Universal RNA Reagent Kit for lon Torrent Platform IFU AK0025 8 Rev B ARCHER E by enzymatics Step 4 End Repair dA Tailing 4 1 Gently open the End Repair dA Tailing SA0004 foil pouch by tearing along the indents located at the top of the silver package 4 2 Remove the blue 8 tube strip Each tube in the strip provides a single reaction 4 3 Centrifuge briefly to ensure lyophilized material is in the bottom of the tube 4 4 f you would like to use fewer than eight reactions detach the appropriate number of tubes carefully using clean scissors or a new razor blade Store the remaining unused tubes in the sealed foil pouch with the desiccant provided at 4 C 4 5 Transfer 40 uL of the Second Strand cDNA Synthesis reaction Step 3 7 into tube containing lyophilized End Repair dA Tailing SA0004 reagents and mix well by pipetting up and down 6 8 times Spin briefly to collect contents at the bottom of the tube 4 6 Incubate the reaction in a thermal cycler with a heated lid set to gt 100 C and incubate as follows Incubation Incubation otep Temperature Time 1 1 C 15 min 2 947 15 15 min 3 JOA 15 min 4 4 C Hold 4 7 Ensure the reaction cools to 4 C and briefly centrifuge End Repair reaction before proceeding 4 8 Gently open a pouch of Archer MBC Adapters for lon Torrent by tearing along the indents located at
9. from left to right as shown below Be sure the label is placed where it Will not be compromised when placed in a thermal cycler 10 Archer Universal RNA Reagent Kit for lon Torrent Platform IFU AK0025 8 Rev B ARCHER gt by enzymatics roy 4 79 74b 7 1 Centrifuge briefly to ensure lyophilized material is in the bottom of the tube If you would like to use fewer than eight reactions detach the appropriate number of tubes carefully using clean scissors or a new razor blade Store the remaining unused tubes in the sealed foil pouch with the desiccant provided at 4 C To the Second PCR tube on ice add 5 1 Purified library DNA Step 6 17 18 uL Liquid GSP2 Mix 2 uL Total 20 uL Allow the pellet to dissolve and then pipet up and down 6 8 times to mix Spin briefly to collect contents at the bottom of the tube Incubate the reaction as follows Note the ramp rate between 98 C and 68 C consult your instrument user s manual to confirm that this setting is correct Ensure the lid temperature tracks 5 C above the incubation temperature or set the lid to 100 C Incubation Temperature Incubation Time of cycles sia IC 30 sec 1 98 C 10 sec 24 68 C ramp rate of 2 3 C sec 30 sec TER D min 1 4 C HOLD 1 NOTE The number of unique molecules will be reduced when the PCR cycles are increased and can be decreased based on user experience with different amount of input materia
10. iate number of tubes carefully using clean scissors or a new razor blade Store the remaining unused tubes in the sealed foil pouch at 4 C 65 To the First PCR tube on ice add Purified library DNA Step 5 16 18 uL Liquid GSP1 Mix 2 uL Total 20 uL 6 6 Allow the pellet to dissolve and then pipet up and down 6 8 times to mix Spin briefly to collect contents at the bottom of the tube 6 7 Incubate the reaction as follows Note the ramp rate between 98 C and 68 C consult your instrument user s manual to confirm that this setting is correct Ensure the lid temperature tracks 5 C above the incubation temperature or set the lid to gt 100 C Incubation Incubation Temperature Time of cycles 98 C 30 sec 1 J8 C 10 sec 90 68 C ramp rate of 2 3 C sec 30 sec 7250 3 min 1 4 C HOLD 1 9 Archer Universal RNA Reagent Kit for lon Torrent Platform IFU AK0025 8 Rev B ARCHER gt by enzymatics NOTE If library yields are too low the cycle number can be increased up to 22 cycles The number of unique molecules will be reduced when the PCR cycles are increased and can be decreased based on user experience with different amount of input material and specific sample types Post First PCR AMPure XP Beads Purification 6 8 Refer to manufacturer s protocol for details on methods of purification 6 9 Add 16 uL of AMPure XP beads to the 20 ul reaction for a ratio of 0 8X 6 10 Vortex well or pipette 10 times
11. l and specific sample types Post Second PCR AMPure XP Beads Purification 7 8 7 9 Refer to manufacturer s protocol for details on methods of purification Add 16 uL of AMPure XP beads to the reaction for a ratio of 0 8X 7 10 Vortex well or pipette 10 times to mix and incubate for 5 minutes at room temperature 7 11 Collect beads with magnet for 2 4 minutes or until solution is clear 7 12 Carefully pipette off and discard supernatant without disturbing the beads 7 13 Wash twice with 200 uL of 70 ethanol while on the magnet Spin down and carefully remove remaining supernatant while taking care not to resuspend beads 7 14 After the second wash dry beads at room temperature for b minutes 7 15 Elute cDNA in 24 uL of 10 mM Tris HCI Remove tubes from the magnet and thoroughly resuspend the beads with the 10 mM Tris HCI 7 16 Place cDNA bead solution back on magnet for 2 minutes 11 Archer Universal RNA Reagent Kit for lon Torrent Platform IFU AK0025 8 Rev B ARCHER gt by enzymatics ct AW 7 17 Carefully transfer 24 uL of the purified cDNA solution to a fresh 200 uL PCR tube or proceed directly to otep 8 Be sure to avoid transferring beads to the fresh tube Stopping point It is OK to stop and store the library at 20 C otep 8 Quantify Library and Sequence 8 1 8 2 0 9 Use the KAPA Biosystems qPCR kit KK4827 for lon Torrent to quantify the concentrati
12. ntents at the bottom of the tube 4 14 Immediately proceed to Step 5 otep 5 Adapter Ligation 5 1 Gently open the Adapter Ligation SA0005 foil pouch by tearing along the indents located at the top of the silver package 5 2 Remove the red 8 tube strip Each tube in the strip provides a single reaction 5 3 Centrifuge briefly to ensure lyophilized material is in the bottom of the tube 9 4 f you would like to use fewer than eight reactions detach the appropriate number of tubes carefully using clean scissors or a new razor blade Store the remaining unused tubes in the sealed foil pouch with the desiccant provided at 4 C 5 5 Transfer 50 uL of the End Repaired dA tailed DNA with the annealed lon Torrent MBC Adapters Step 4 13 into the tube containing Adapter Ligation mix Allow pellet to dissolve and then pipet up and down 6 8 times to mix Spin briefly to collect contents at the bottom of the tube 5 6 Incubate the reaction as follows If a thermal cycler is used either set the thermal cycler lid to off or leave it open during the incubation Incubation Incubation otep Temperature Time 1 Mace 30 min 2 22 C 30 min Post Ligation AMPure XP Beads Purification 5 7 Refer to manufacturer s protocol for details on methods of purification 5 8 Add 40 uL of AMPure XP beads to the 50 uL reaction for a ratio of 0 8X 5 9 Vortex well or pipette 10 times to mix and incubate for 5 minutes at room temperature 5 10
13. om Priming mixture Step 1 9 to the lyophilized First Strand cDNA Synthesis pellet and mix well by pipetting up and down spin briefly to collect contents at the bottom of the tube 2 6 Place the tubes into a thermal cycler with a heated lid set to gt 100 C and incubate as follows Incubation Incubation step Temperature Time 1 25 C 10 min 2 42 C 30 min 3 80 C 20 min 4 4 C Hold 2 7 Remove the PCR tubes from the thermal cycler and place on ice 3 1 Gently open the Second Strand cDNA Synthesis SA0003 foil pouch by tearing along the indents located at the top of the silver package 3 2 Remove the yellow 8 tube strip Each tube in the strip provides a single reaction 3 3 Centrifuge briefly to ensure lyophilized material is in the bottom of the tube 3 4 f you would like to use fewer than eight reactions detach the appropriate number of tubes carefully using clean scissors or a new razor blade Store the remaining unused tubes in the sealed foil pouch with the desiccant provided at 4 C 3 5 Tothe Second Strand Synthesis tube on ice add Ultra Pure Water SA0021 20 uL First Strand cDNA Synthesis reaction Step 2 7 20 uL Total 40 uL 3 6 Mix well by pipetting gently up and down 6 8 times Spin briefly to collect contents at the bottom of the tube 3 7 Incubate at 16 C for 1 hour If a thermal cycler is used for the incubation do not use a heated lid or close the heated lid Do not allow the temperature to rise
14. on of each library Assume a 250 bp fragment length After quantification pool the barcoded libraries at equimolar concentrations and perform template preparation following the manufacture s protocol Sequence on an lon Torrent PGM 318 chip A generic sequencing template can be created on the Torrent Server after uploading the index tag files and this template can be used to plan the run In order to maintain the appropriate read coverage per target it is suggested to limit input to 10 samples per 318 chip 8 1 1 Samples within the pool should be demulitplexed using the Torrent Server with the appropriate barcode sequence In addition the index tag file can be downloaded from our site for convenience http www enzymatics com archer This assay workflow leads to constructs with highly efficient ePCR amplification during PGM template preparation In order to achieve 10 30 unenriched template positive ISPs library should be loaded into the ePCR at 13 pM using the lon Torrent PGM Template OT2 200 kit Typically lon Torrent recommends using 20 pM of library into ePCR However with our more efficient amplified constructs loading at a lower concentration is necessary to avoid a high percentage of polyclonal ISPs After the first chip the loading concentration can be adjusted accordingly to achieve the optimal percentage of unenriched template positive ISPs Upon completion of the run the data should be analyzed using the Archer
15. s embedded in the lon Torrent Barcode Adapters In order to maintain appropriate coverage depth it is recommended to cap each PGM run at 2 10 samples per 318 chip In general larger panels with more targets will require higher sequencing coverage depth and should be run with fewer samples per chip Below are some recommendations for panels of different sizes Archer Panel of Targets Assay Recommended of samples 318 Chip FusionPlex ALK RET ROS1 Panel v2 29 7 10 FusionPlex Heme Panel 132 2 3 FusionPlex Sarcoma Panel 134 2 3 Input Nucleic Acid Concentration and Purification e Total nucleic acid is the preferred input for this assay e DO NOT treat the extracted total nucleic acid with DNase as this will critically reduce the quality of RNA in the sample e f nucleic acid is from FFPE tissue it is recommended to use Agencourt FormaPure A33342 for extraction e When possible it is recommended to increase the total nucleic acid input which will increase library complexity and improve the sensitivity of the assay If higher library complexity is desired the assay can tolerate up to 250 ng of total nucleic acid e The minimum recommended input for the assay is 20 ng of total nucleic acid Alternatively 10 ng of RNA may be used e Efficient library preparation can be achieved with as little as 2 ng of total nucleic acid provided that the starting material is of high quality and is not degraded However reduced inp
16. the top of the silver package 49 Remove the clear 8 tube strip from the foil pouch Each tube in the strip provides a single reaction and each tube contains a different lon Torrent MBC Adapter or lon Torrent Barcode Adapter For example reactions 1 through 8 correspond to MBC Adapters 1 through 8 4 9 1 CRITICAL Upon removing the 8 tube strip from the pouch position the tubes with the hinges to the back and use a permanent marker to label the tubes 1 through 8 from left to right as shown below Be sure to label and track the index number added to each sample from this point forward 4 10 Centrifuge briefly to ensure lyophilized material is in the bottom of the tube 4 11 If you would like to use fewer than eight reactions detach the appropriate number of tubes carefully using clean scissors or a new razor blade Store the remaining unused tubes in the sealed foil pouch with 7 Archer Universal RNA Reagent Kit for lon Torrent Platform IFU AK0025 8 Rev B ARCHER gt by enzymatics As Y ae 7 A cw id the desiccant provided at 4 C Be sure to track which indices were used to ensure index compatibility when used in later experiments 4 12 To the Archer MBC Adapters for lon Torrent tube on ice add Ultra Pure Water SA0021 10 uL End Repaired dA tailed DNA Step 4 7 40 uL Total 50 uL 4 13 Allow the pellet to dissolve and then pipet up and down 6 8 times to mix Spin briefly to collect co
17. to mix and incubate for b minutes at room temperature 6 11 Collect beads with magnet for 2 4 minutes or until solution is clear 6 12 Carefully pipette off and discard supernatant without disturbing the beads 6 13 Wash twice with 200 uL of 70 ethanol while on the magnet Spin down and carefully remove remaining supernatant while taking care to not resuspend beads 6 14 After the second wash dry beads at room temperature for 5 minutes 6 15 Elute cDNA in 24 uL of 10 mM Tris HCl Remove tubes from the magnet and thoroughly resuspend the beads with the 10 mM Tris HCI 6 16 Place cDNA bead solution back on magnet for 2 minutes 6 17 Carefully transfer 22 uL of the purified library solution to a fresh 200 uL PCR tube or proceed directly to Step 7 Be sure to avoid transferring beads to the fresh tube Stopping point It is OK to stop and store the library at 20 C Step 7 Second PCR NOTE The Archer Universal RNA Reagent Kits for lon Torrent Platform kits do not contain gene specific primers GSPs in the reaction pellet 7 1 Gently open the Second PCR SA0015 foil pouch by tearing along the indents located at the top of the Silver package 7 2 Remove the clear 8 tube strip from the foil pouch Each tube in the strip provides a single reaction 72 1 CRITICAL Upon removing the 8 tube strip from the pouch position the tubes with the hinges to the back and use a permanent marker to label the tubes 1 through 8
18. ut will decrease library complexity due to the restricted amount of starting unique target molecules When using less than 10 ng of input material the PCR cycling conditions Steps 6 and 7 may need to be altered e The use of EDIA containing buffers in this protocol may result in lower library yields Be sure to use buffers that do not contain EDTA i e use Tris HCl and not Tris EDTA buffer 4 Archer Universal RNA Reagent Kit for lon Torrent Platform IFU AK0025 8 Rev B ARCHER by enzymatics Before You Begin Make fresh 10 mM Tris HCl o Mix 20 uL 500 mM Tris HCl pH 8 0 840020 with 980 uL Ultra Pure Water 840021 Make fresh 70 ethanol o Add 14 mL 100 ethanol ACS grade not included to entire bottle containing Ultra Pure Water for Ethanol Dilution SA0022 o Note the date on which ethanol is added 70 ethanol is appropriate for use for one week after mixing When not in use tightly close the bottle cap to ensure minimal evaporation Instructions for Use otep 1 Random Priming L4 1 2 1 9 1 4 19 1 6 ie 1 8 1 9 Pre heat thermal cycler to 65 C with a heated lid Gently open the Random Priming SA0001 foil pouch by tearing along the indents located at the top of the silver package Remove the green 8 tube strip Each tube in the strip provides a single reaction Centrifuge briefly to ensure lyophilized material is in the bottom of the tube If you would like to use fewer than eight
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