IFU-AK0024-8 Archer Universal RNA Reagent Kit
Contents
1. UT i i NU Y gt S Instructions for Use ARCHER by enzymatics Archer Universal RNA Reagent Kit for IIlumina Platform AK0024 8 Table of Contents Archer Universal RNA Reagent Kit for Illumina Platform e 1 TEOT Pont ERR ne ey nS EO ne ne ne ney eer rere ee eee ne eee ney eee eee 1 re i010 Ue Moo C C 2 Modular AS aV FONA ee ea ema Pf E Pn Pet E eee ee eee ee 2 OE diu E anatece 2 Version Additions and D OE BEL ossoiteneseengset uta eitiutetuc Ris Deut tesi colitur mde toto custo diocesan elg SCENDE USD 3 Sui nig one ee E re ee E ee ne ee ee 3 Materials Required But Not Supplied uo ccc cssecsescesssecsescesessccessesessscessseessseessseesssecsessssssecseseeaesecsesseaesecsesseseseceenees 3 Eis nisi aU Tr Ue Ue UNO S REP E TET 3 vin C O M 4 Sample MOIpIEXINE V 4 Ba oe DE O ee E E E E E E E E E 4 Input Nucleic Acid Concentration and Purification s0010n0n0nenn010nononsnenneransnsnsnrnraransrsnsnrnrararsnsnsntnrararsrsrsnenrarnrersrerennns 4 Pe oe OU DEEI r EN E E E E E E E R aera eee ee D HU COn Or UE e E eects E EE E E EA S D Sz NEM c neu mE 9 Step 2 First Strand cDNA Synthesis sasicsn caves topwasinianoh penses tt btte tarte aea demas ennadat2. 7 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 7 5 Tothe Second PCR tube on ice add Veen Purified library DNA Step 6 17 18 uL Liquid GSP2 Mix 2 uL Total 20 uL 7 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 7 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 Temperature Incubation Time of cycles 98 C 30 sec il 98 C 10 sec 24 68 C ramp rate of 2 3 C sec 30 sec PE 3 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 material and specific sample types Post Second PCR AMPure XP Beads Purification 7 8 Refer to manufacturer s protocol for details on methods of purification 7 9 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 b minutes at room temperature 7 11 Collect beads with magnet for 2 4 minutes
3. Primers 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 4 Archer Universal RNA Reagent Kit for lllumina Platform IFU AK0024 8 Rev B ARCHER E by ii ida S KA qS x EN m 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 input 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 EDTA 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 Before Yo
4. and place on ice for 2 minutes then briefly centrifuge before proceeding with First Strand cDNA Synthesis D Archer Universal RNA Reagent Kit for Illumina Platform IFU AK0024 8 Rev B ARCHER by enzymatics otep 2 First Strand cDNA Synthesis 2 1 Gently open the First Strand cDNA Synthesis 840002 foil pouch by tearing along the indents located at the top of the silver package 2 2 Remove the purple 8 tube strip Each tube in the strip provides a single reaction 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 Random Priming mixture Step 1 9 to the lyophilized First Strand cDNA Synthesis pellet and mix well by pipetting up and down opin 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 2515 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 ind
5. dA Tailing 40 uL Transfer 10 uL H20 We Veo We he MBC Adapters Adapter Ligation 50 uL Transfer e e o o o o o o 20 uL Transfer First PCR EP e v e v9 9 9 9 20 uL Transfer Purification Peg 20 uL Transfer SA ng TH 20 uL Transfer _ 24 uL Transfer C Quantitate Library and Sequence 2 Archer Universal RNA Reagent Kit for IIlumina Platform IFU AK0024 8 Rev B ARCHER by enzymatics Version Additions amp Changes e Added multiplexing recommendations for the Archer FusionPlex Heme and Sarcoma Panels e Instructions included for the preparation of PhiX to achieve a final concentration of 10 pM in 0 2 N NaOH starting from a 10 nM stock solution Kit Contents 500 mM Tris HCl pH 8 0 SA0020 Ultra Pure Water SAQ021 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 SA0009 Step 7 Second PCR 840013 pe DU peg go oolosc Materials Required But Not Supplied l Archer MBC Adapters for lllumina Archer FusionPlex Assay Cat AK0028 8 AK0029 8 AK0032 8 Agencourt AMPure XP Beads Cat 463881 Life Technologies DynaMag Cat 12331D 100 ethanol ACS grade KAPA Biosystems Library Quantification Kit IIlumina
6. or until solution is clear 7 12 Carefully pipette off and discard supernatant without disturbing the beads 11 Archer Universal RNA Reagent Kit for IIlumina Platform IFU AK0024 8 Rev B ARCHER by enzymatics gt p EET B 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 HCl 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 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 Use the KAPA Biosystems qPCR kit KK4824 for Illumina to quantitate the concentration of each library Assume a 250 bp fragment length After quantification pool the barcoded libraries at equimolar concentrations and sequence on an lllumina 9 MiSeq It is recommended to cap each MiSeg run at 48 samples to maintain appropriate coverage depth 8 2 Run the MiSeqQ using the read level sequence in the table below In addition a reference sample sheet is available for download at http www e
7. IFU AK0024 8 Rev B ARCHER by enzymatics Limitations of Use For Research Use Only Not for use in diagnostic procedures This 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 Illumina 9 and MiSeg are registered trademarks of Illumina Inc Agencourt AMPure and FormaPure are registered trademarks of Agencourt Biosciences Corporation a Beckman Coulter company Life Technologies and DynaMag are trademarks of Thermo Fisher Scientific Inc 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 Enzymatics Inc e enzymatics E 5 P4 Phone 888 927 7027 220 14 Archer Universal RNA Reagent Kit for IIlumina Platform IFU AK0024 8 Rev B
8. Universal Cat KK4824 Custom Primer Panels designed at http assay enzymatics com If nucleic acid is from FFPE tissue it is recommended to use Agencourt FormaPure Cat A33342 for extraction So Ge a ae 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 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 IIlumina Platform IFU AK0024 8 Rev B ARCHER by enzymatics Storage All components of the Archer Universal RNA Reagent Kit Part AK0024 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 MiSeq multiple samples should be sequenced simultaneously Samples can be identified through a unique nucleotide sequence that is part of the adapter attached to the n
9. ch sample from this point forward 58 6 1 7 Archer Universal RNA Reagent Kit for IIlumina Platform IFU AK0024 8 Rev B ARCHER E by enzymatics gt gt s B ENT ARR 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 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 tube for IIlumina 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 contents at the bottom of the tube 4 14 Immediately proceed to Step 5 otep b 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 ra
10. e 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 WAG 15 min 2 37 C 15 min 3 OG 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 Illumina by tearing along the indents located at the top of the silver package 4 9 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 Illumina amp MBC 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 ea
11. ents 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 cDNA 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 b Archer Universal RNA Reagent Kit for IIlumina Platform IFU AK0024 8 Rev B ARCHER E by enzymatics P AN a ENET E 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 above 16 C Stopping point It is OK to stop and store the library at 20 C 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 th
12. f 10 mM Tris HCl Remove tubes from the magnet and thoroughly resuspend the beads with the 10 mM Tris HCI Place cDNA bead solution back on magnet for 2 minutes 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 Illumina Platform do not contain gene specific primers GSPs in the reaction pellet 1 iz 10 Gently open the Second PCR SA0013 foil pouch by tearing along the indents located at the top of the Silver package 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 MiSeq Index 1 Barcode Primer 1 through 8 Reactions 1 through 8 correspond to MiSeq Index 1 through 8 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 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 Archer Universal RNA Reagent Kit for IIlumina Platform IFU AK0024 8 Rev B ARCHER by enzymatics 7 3 Centrifuge briefly to ensure lyophilized material is in the bottom of the tube
13. for 5 minutes 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 Place cDNA bead solution back on magnet for 2 minutes 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 otep b First PCR NOTE The Archer Universal RNA Reagent Kits do not contain gene specific primers GSPs in the reaction pellet 6 1 6 2 SN 6 4 6 5 6 6 6 7 Gently open the First PCR SA0009 foil pouch by tearing along the indents located at the top of the silver package Remove the clear 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 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 at 4 C To the First PCR tube on ice add Purified library DNA Step 5 16 18 uL Liquid GSP1 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 ins
14. ibrary pool 10 uL 0 2 N NaOHin 1 5 mL microcentrifuge tube vortex briefly 8 5 2 Incubate for 5 min at room temperature 8 5 3 Add 980 uL ice cold Hyb buffer this comes with the MiSeq cartridge 8 5 4 This makes a 20 pM stock vortex briefly to mix 8 5 5 Mix 500 uL Hyb Buffer 500 uL 20 pM library in new 1 5 mL microcentrifuge tube and vortex briefly 10 pM Library stock 8 5 6 Mix 900 uL 10 pM library stock 100 uL 10 pM denatured PhiX and vortex briefly 8 5 7 This creates the final loading pool of 10 pM 10 PhiX 8 5 8 Add the entire 1000 uL to the MiSeq cartridge and start the run 8 6 Starting from a 4 nM pool 8 6 1 Mix 10 uL 4 nM library pool 10 uL 0 2 N NaOQHin 1 5 mL microcentrifuge tube vortex briefly 8 6 2 Incubate for 5 min at room temperature 8 6 3 Add 980 uL ice cold Hyb buffer this comes with the MiSeq cartridge 8 6 4 This makes a 40 pM stock vortex briefly to mix 8 6 5 Mix 750 uL Hyb Buffer 250 uL 40 pM library in new 1 5 ml microcentrifuge tube and vortex briefly 10 pM Library stock 8 6 6 Mix 900 uL 10pM library stock 100 uL 10 pM denatured PhiX and vortex briefly 8 6 7 This creates the final loading pool of 10 pM 10 PhiX 8 6 8 Add the entire 1000 uL to the MiSeq cartridge and start the run 8 7 Upon completion of the run the data should be analyzed using the Archer Analysis Pipeline http archer enzymatics com 13 Archer Universal RNA Reagent Kit for Illumina Platform
15. nzymatics com archer The reference sample sheet can be modified with the appropriate index sequence tags and reagent tag and uploaded to the MiSeq 9 for the run Level Read Length R1 Read 1 Lol R2 Index Read 1 8 R3 Index Read 2 8 R4 Read 2 1541 8 3 Load library on Illumina MiSeq at 10 pM with 10 PhiX using the Miseg v2 300 cycle reagent kit following the loading conditions below 8 4 Dilute and denature PhiX to 10 pM in 0 2 N NaOH starting from the 10 nM stock If PhiX has already been diluted skip to step 8 5 or 8 6 Additionally dilution and denaturation of PhiX can be performed concurrently with the 2 or 4 nM pooled library sample 84 1 Mix2 uL of 10 nM PhiX stock 8 uL of Ultra Pure Water SA0021 in a 1 5 mL microcentrifuge tube to dilute LOnM PhiX stock to 2nM 842 Mix 10 uL of the 2 nM PhiX stock 10 uL of 0 2 N NaOH in a 1 5 mL microcentrifuge tube vortex briefly 8 4 3 Incubate for b minutes at room temperature 844 Add 980 uL ice cold Hyb buffer this comes with the MiSeq cartridge 845 This makes a 20 pM stock vortex briefly to mix 8 4 6 Mix 500 uL Hyb Buffer 500 uL 20 pM PhiX in a new 1 5 mL microcentrifuge tube 12 Archer Universal RNA Reagent Kit for lllumina Platform IFU AK0024 8 Rev B ARCHER by enzymatics 847 This is the final 10 pM working stock of PhiX store at 20 C for a maximum of 2 months 8 5 Starting from a 2 nM pool 8 5 1 Mix 10 uL 2 nM l
16. ote oet oun ng 6 DID paar to 616 C10 OU e D EE E ene en eon ea E MI EM MEM Ca Te 6 Scu Mz NA VEI 7 vc TE 6182 ay 1d 61h 8 Succ ad OY nT ee Yn A Ene g SE Ago HIT RR 10 Sc ay Library and OO GIO E 12 For more information please visit http www enzymatics com archer ssssssssssens 14 1 Archer Universal RNA Reagent Kit for lllumina Platform IFU AK0024 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 Archer Universal RNA Reagent Kit used in conjunction with Archer Assays and MBC Adapters allows users to construct Illumina MiSeq ready libraries from total nucleic acid or RNA samples Universal RNA Reagent Kit FusionPlex Assays MBC Adapters For Research Use Only Not for use in diagnostic procedures Workflow Overview 20 uL of TNA RNA H O Random Priming e eoe o o o oe 0e 906 First Strand cDNA Synthesis 20 uL Transfer 20 uL Transfer 20 uL H20 40 uL Transfer End Repair
17. trument 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 Archer Universal RNA Reagent Kit for IIlumina Platform IFU AK0024 8 Rev B ARCHER by enzymatics 98 C 30 sec 1 98 C 10 sec 20 68 C ramp rate of 2 3 C sec 30 sec E 3 min 1 4 C HOLD 1 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 Doe 6 9 6 10 6 11 6 12 6 13 6 14 6 15 6 16 6 17 Refer to manufacturer s protocol for details on methods of purification Add 16 uL of AMPure XP beads to the 20 uL reaction for a ratio of 0 8X Vortex well or pipette 10 times to mix and incubate for 5 minutes at room temperature Collect beads with magnet for 2 4 minutes or until solution is clear Carefully pipette off and discard supernatant without disturbing the beads 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 After the second wash dry beads at room temperature for 5 minutes Elute cDNA in 24 uL o
18. u Begin e 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 e 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 Step 1 Random Priming 1 1 Pre heat thermal cycler to 65 C with a heated lid 1 2 Gently open the Random Priming SA0001 foil pouch by tearing along the indents located at the top of the silver package 1 3 Remove the green 8 tube strip Each tube in the strip provides a single reaction 1 4 Centrifuge briefly to ensure lyophilized material is in the bottom of the tube 1 5 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 desiccant provided at 4 C 1 6 Place the tubes on ice and to each add Ultra Pure Water SA0021 20 XuL Purified Total Nucleic Acid X uL Total 20 uL 1 7 After the lyophilized pellet dissolves gently pipet up and down 6 8 times and briefly spin down 1 8 Transfer the tubes from ice to the thermal cycler and incubate at 65 C for 5 minutes 1 9 Remove tubes from thermal cycler
19. ucleic 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 kits for Illumina utilize 2 indices in combination to distinguish between samples The first index is added just before Step 5 Adapter Ligation and is embedded in the Archer MBC Adapters for Illumina 9 The second index is added in Step 7 Second PCR and is embedded in MiSeq Index 1 Primers within the Second PCR reaction In order to maintain appropriate coverage depth it is recommended to cap each MiSeg run at 30 48 samples per lane In general larger panels with more targets will require higher sequencing coverage depth and should be run with fewer samples per sample Below are some recommendations for panels of different sizes Archer Panel of Targets Assay Recommended of Samples Lane FusionPlex ALK RET ROS1 Panel v2 29 30 40 FusionPlex Heme Panel 132 20 30 FusionPlex Sarcoma Panel 134 20 30 Barcode Diversity The Illumina MiSeq will work best when index diversity within a run is high For example if eight samples are included in a run and the user chooses to use only one MBC Adapter paired with eight different MiSeq Index 1 Primers the run may fail due to low barcode diversity In this example it is best to use eight different Archer MBC Adapters paired with eight different MiSeq Index 1
20. zor 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 Illumina 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 16 C 30 min 2 2S 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 Collect beads with magnet for 2 4 minutes or until solution is clear 8 Archer Universal RNA Reagent Kit for Illumina Platform IFU AK0024 8 Rev B ARCHER by enzymatics P 5 11 Dus Duo 5 14 5 15 5 16 gt s EE A ENT l Carefully pipette off and discard supernatant without disturbing the beads 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 After the second wash dry beads at room temperature
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