IFU-AK0042-8 Archer Universal RNA Reagent Kit v2 for Ion Torrent
Contents
1. a N ARCHER ae Archer Universal RNA Reagent Kit v2 for lon Torrent Platform 8 AK0042 8 Table of Contents IER ln 1 Keser eee sees 2 tele lee E le s 2 Modular Agsavkormmgt c caccetsesescecseseeatsseeneneccersrsereunnacaeterseceneagastaneeataeagestsaseqetadgeshpnancanadgestetenatabderstsassuenedserapsattpieaencssnee 2 KIT CONTENTS eet ne sda eS ded ede sete ee ed ern ed cere 2 Materials Required But Not Supplied oo ccc csssscssssesssscsesnesesscsesssesessscessscesssssussssssnssssscssasesescsseseaseseeceseseesescanesescens 5 General PRECAUTIONS geed Ate er A Ah ed eee ee 5 Storage and Hamlin es cscccee de reecth he aia EES eee ete eee ee S 5 Sample MUItiPlexig eee eeecceceeseceessesseesesecsessessesseseessescsnssessesscsessecscsnsesssesesueesesesseescssssessesussesanseeseeessesseesssesseteesseeeeaneeeeeees 6 Input Nucleic Acid Concentration and PUrifiCation eee ccceseseesessesseesesessecseseeseesessesteasseeseeseseeseesssessteseeeeeeesneeeeeees 6 Archer PreSeg RNA QC Aesgy reeet 7 BG Ee Ee 7 PROUOCOM EE 8 Step 1 Random Drummg rarnana rinan nnnrna aranan ann 8 otep 2 First Strand CDNA SYnth s 8 nennisnan rensar nna Ea Ea EA E R ERE E 8 SP 3 Prese RNA UE AS E H Step 4 Second geleet 10 SP3 Eig ENGI GAN Ite cds carte S A E 10 Step 6 Adapter LIGATION ceseccecccsesessesseceseeseesessessesessessssnssecsnesssecsscsesssssacssssucesssesuesassssecsssasssanssessseassesreeessesseeassaesteansaeees 12 OLD ZE FISERCR scot2. curve setting Add 21 ul water to 19 ul First Strand cDNA Incubation EE EE EE Synthesis mixture and then transfer to Temperature Time Second Strand cDNA Synthesis reagent eg est 1 Incubate as indicated with unheated lid Optional stopping point after this step Store at 20 C First Strand cDNA Synthesis Lo GC ov tagg ony kel Car OAR ox GES ns 3 Archer Universal RNA Reagent Kit v2 for lon Torrent Platform 8 IFU AK0042 8 Rev C S ARCHER A End Repair Adapter dA Tailing Ligation Transfer 40 ul Second Strand cDNA incubation Synthesis mixture to End Repair dA Tailing reagent Incubate as indicated 72 C AMPure XP clean up 2 5x Elute in 52 pl Incubation Incubation Temperature Time Incubate as indicated Pp 2 JL 22 80min with unheated lid AMPure XP clean up 1 2x Elute in 20 pl Transfer 50 ul End Repair dA Tailing mixture to MBC Adapters Then mix and transfer to Adapter Ligation reagent al Optional stopping point after this step Store at 20 C Transfer 18 ul purified Adapter Ligation mix Incubation Incubation DE PR PCR reagent Then mix and add Temperature Time y g ege Incubate as indicated e See 65 C 60 panel specific ramp rate 0 28 C sec sec protocol Optional topping pin Pec amin 1 after this step Store at 20 C AMPure XP clean up 1 2x Elute in 20 pl Transfer 18 ul purified First PCR mixture to Incuba
3. C 15 min 3 72 C 15 min 4 A C Hold 10 Archer Universal RNA Reagent Kit v2 for lon Torrent Platform 8 IFU AK0042 8 Rev C s E ARCHER TO a 3 43 WW 5 6 Ensure the reaction cools to 4 C and briefly centrifuge reactions before proceeding Post End Repair dA Tailing AMPure XP Beads Purification 5 7 Add 100uL of AMPure beads to the 40uL reaction for a ratio of 2 5X Make sure that the AMPure XP beads have reached room temperature and are fully resuspended by vortexing prior to adding to the sample 5 8 Vortex well or pipette 10 times to mix and visually inspect the color of the sample to ensure a homogenous mixture 5 9 Incubate for 5 minutes at room temperature Place tubes on magnet for 2 4 minutes or until beads are pelleted and solution is clear 5 10 Carefully pipette off and discard supernatant without disturbing the beads 5 11 Without disturbing the separated beads wash by adding 200 uL of 70 ethanol while on the magnet and incubate for 30 sec at room temperature Carefully remove ethanol and discard Repeat this procedure for a total of 2 washes 5 12 After the final wash briefly spin down and carefully remove remaining ethanol supematant while taking care not to disturb the beads With the caps open dry beads at room temperature for 5 minutes Take care not to over dry the beads as this can reduce yield 5 13 Remove tubes from the magnet and elute DNA by thoroughly resuspending the beads in 52 uL of Ul
4. Step 6 11 Archer Universal RNA Reagent Kit v2 for lon Torrent Platform 8 IFU AK0042 8 Rev C S ARCHER a Step 6 Adapter Ligation 6 1 Gently open the Adapter Ligation SA0005 foil pouch by tearing along the indents located at the top of the silver package 6 2 Remove the red 8 tube strip Each tube in the strip provides a single reaction 6 2 1 If you would like to use fewer than 8 reactions carefully detach the appropriate number of tubes using clean scissors or a new razor blade Store the remaining unused tubes at 4 C in the sealed foil pouch with the provided desiccant 6 3 Centrifuge briefly to ensure lyophilized material is in the bottom of the tube 6 4 Transfer 50 uL of the End Repaired dA tailed DNA with the annealed lon Torrent MBC Adapters Step 5 19 into the tube containing Adapter Ligation mix Allow pellet to dissolve and then gently pipet up and down 6 8 times to mix Spin briefly to collect contents at the bottom of the tube 6 5 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 Step Temperature Time 1 16 C 30 min 2 22 C 30 min 3 4 C Hold Post Ligation AMPure XP Beads Purification 6 6 Add 60 uL of AMPure XP beads to the 50 uL reaction for a ratio of 1 2X Make sure that AMPure XP Beads have reached room temperature and are fully resuspended by vortexing prior to a
5. the AMPure bead solution back on magnet for 2 minutes Carefully transfer 18 uL of the purified solution to a fresh 200 uL PCR tube or proceed directly to Step 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 Step 8 Second PCR NOTE The Archer Universal RNA Reagent Kits v2 do not contain gene specific primers GSPs in the reaction pellet Please use the Archer FusionPlex or custom assay GSPs 8 1 8 2 8 3 84 8 5 8 6 14 Gently open the Second PCR SA0112 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 8 2 1 If you would like to use fewer than 8 reactions carefully detach the appropriate number of tubes using clean scissors or a new razor blade Store the remaining unused tubes at 4 C in the sealed foil pouch with the provided desiccant Centrifuge briefly to ensure lyophilized material is in the bottom of the tube To the Second PCR tube on ice add Purified library DNA Step 7 16 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 95 C and 65 C consult your instrument user s manual to confir
6. to collect contents at the bottom of the tube Place the tubes into a thermal cycler with a heated lid set to gt 100 C and incubate as follows Archer Universal RNA Reagent Kit v2 for Ion Torrent Platform 8 IFU AK0042 8 Rev C CH ARCHER a Step Incubation Temperature Incubation Time 1 25 C 10 min 2 42 C 30 min 3 80 C 20 min 4 4 C Hold 2 6 Remove the PCR tubes from the thermal cycler briefly centrifuge and place on ice Step 3 PreSeq RNA QC Assay 3 1 Thaw 10x VCP Primer Mix SA0121 at room temperature 3 2 Transfer 1 uL of cDNA from First Strand cDNA synthesis to a new PCR tube containing 9 uL Ultra Pure Water SA0021 3 3 Briefly vortex and centrifuge diluted cDNA from Step 3 2 3 4 For 1 Reaction setup the qPCR assay as follows Components For a 10 uL Reaction 2X SYBR Green Master Mix Du Diluted cDNA sample AuL 10X VCP Primer Mix 1uL Total 10uL NOTE Master mix preparation is recommended to avoid variability due to pipetting small volumes Create a volume of master mix comprising 2X SYBR Green Master Mix and 10X VCP Primer Mix adequate for the total number of planned reactions Aliquot appropriate volume of master mix into each reaction tube then add appropriate volume of diluted cDNA sample 3 5 Place the reactions in a real time PCR instrument and perform cycling with the following protocol Step Incubation Temperature Incubation Time of cycles Activation Denatura
7. 6 Archer Universal RNA Reagent Kit v2 for lon Torrent Platform 8 IFU AK0042 8 Rev C CH ARCHER a Archer PreSeg RNA QC Assay The Archer PreSeq RNA QC Assay is used to provide data about the integrity of amplifiable RNA in a sample and is predictive of Sequencing success This assay measures the abundance of RNA that is of sufficient length to permit fusion calling Laboratories should determine their own threshold for predicted sample failure based on the PreSeq RNA QC qPCR assay results At Archer we have found that Cq values of 30 cycles or more correspond with sequencing failure due to poor RNA integrity while Cq values under 28 5 tend to correlate with high quality sequencing data 1 0 The receiver operating characteristic curve plot ROC plot on the left demonstrates the sensitivity and 28 589 0 971 0 837 specificity of the PreSeq assay for predicting mean control start sites For this data set of 138 FFPE samples a Cq value of 28 589 is most predictive 97 1 J percent of samples with Cq lower than 28 589 have greater than 10 mean control start sites sensitivity and 83 7 percent of samples with Cq higher than 28 589 had less than 10 mean control start sites specificity Of the 138 samples 47 were known to have J rearrangements by FISH or RT PCR AMP identified all 47 0 6 0 8 Sensitivity 0 4 0 2 0 0 T T T T T T 1 0 0 8 0 6 0 4 0 2 0 0 Specificity The PreSeq RNA QC assay is incorpo
8. GM SYBR Life Technologies and DynaMag are registered trademarks of Thermo Fischer Scientific Inc Bio Rad and iTaq are trademarks of Bio Rad Laboratories 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 aregistered trademark of Molecular Bio Products Inc We 2477 55 Street Suite 202 Boulder CO 80301 16 ArcherDx Inc 303 357 9001 www archerdx com Archer Universal RNA Reagent Kit v2 for lon Torrent Platform 8 IFU AK0042 8 Rev C
9. an be identified through a unique nucleotide sequence that is part of the molecular barcode adapter ligated to the nucleic acid sequence of interest 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 v2 for lon Torrent Platform utilizes a single index to distinguish between samples The index is added during Step 6 Adapter Ligation and is embedded in the lon Torrent Barcode Adapters In order to maintain appropriate coverage depth it is recommended that users determine the maximum number of samples that can be run on a 318 chip assuming 5 million reads per run 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 initial recommendations for panels of different sizes Archer Panel of Targets Assay Recommended of Reads FusionPlex ALK RET ROS1 29 500 000 1 000 000 Panel v2 FusionPlex Heme Panel 132 1 000 000 1 500 000 FusionPlex Sarcoma Panel 134 1 000 000 1 500 000 FusionPlex NTRK Panel 17 500 000 1 000 000 FusionPlex FGFR Panel 17 500 000 1 000 000 FusionPlex Solid Tumor Panel 291 2 000 000 3 000 000 FusionPlex Lung Thyroid Panel Al 1 000 000 1 500 000 Input Nucleic Acid Concentration and Purification e Total nucleic acid is the preferred input for this assay e DONOT t
10. d The number of PCR cycles can be decreased based on user experience quantity of input material and specific sample types Post First PCR AMPure XP Beads Purification 7 7 7 8 7 9 13 Add 24 uL of AMPure XP beads to the 20 uL reaction for a ratio of 1 2X Make sure that AMPure XP Beads are fully resuspended by vortexing prior to adding to the sample Vortex well or pipette 10 times to mix and visually inspect the color of the sample to ensure a homogenous mixture Incubate for 5 minutes at room temperature Archer Universal RNA Reagent Kit v2 for lon Torrent Platform 8 IFU AK0042 8 Rev C vi ARCHER ae 7 10 7 11 7 12 7 14 7 15 7 16 a ek Aa Place tubes on magnet for 2 4 minutes or until beads are pelleted and solution is clear Carefully pipette off and discard supernatant without disturbing the beads Without disturbing the separated beads wash by adding 200 ul of 70 ethanol while on the magnet and incubate for 30 sec at room temperature Carefully remove ethanol and discard Repeat this procedure for a total of 2 washes After the final wash briefly spin down and carefully remove remaining supernatant while taking care not to disturb the beads With caps open dry beads at room temperature for 5 minutes Take care not to over dry the beads as this can reduce yield Remove tubes from the magnet and elute DNA by thoroughly resuspending the beads in 20 uL of 10 mM Tris HCl Place
11. d foil pouch with the provided desiccant Centrifuge briefly to ensure lyophilized material is in the bottom of the tube 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 cDNA Synthesis Step 2 First Strand cDNA Synthesis 2 1 Gently open the First Strand cDNA Synthesis SA0002 foil pouch by tearing along the indents located 2 2 2 3 at the top of the silver package Remove the purple 8 tube strip Each tube in the strip provides a single reaction 2 2 1 If you would like to use fewer than 8 reactions carefully detach the appropriate number of tubes carefully using clean scissors or a new razor blade Store the remaining unused tubes at 4 C in the sealed foil pouch with the provided desiccant Centrifuge briefly to ensure lyophilized material is in the bottom of the tube Place the First Strand cDNA Synthesis tubes on ice and transfer 20 uL of the Random Priming mixture Step 1 8 to the lyophilized First Strand cDNA Synthesis pellet After the lyophilized pellet dissolves mix well by gently pipetting up and down 6 8 times Spin briefly
12. dding to the sample 6 7 Vortex well or pipette 10 times to mix and visually inspect the color of the sample to ensure a homogenous mixture 6 8 Incubate for 5 minutes at room temperature 6 9 Place tubes on magnet for 2 4 minutes or until beads are pelleted and solution is clear 6 10 Carefully pipette off and discard supernatant without disturbing the beads 6 11 Without disturbing the separated beads wash by adding 200 uL of 70 ethanol while on the magnet and incubate for 30 sec at room temperature Carefully remove ethanol and discard Repeat this procedure for a total of 2 washes 6 12 After the final wash briefly spin down and carefully remove remaining supernatant while taking care not to disturb the beads With the caps open dry beads at room temperature for 5 minutes Take care not to over dry the beads as this can reduce yield 6 13 Remove tubes from the magnet and elute DNA by thoroughly resuspending the beads in 20 uL of 10 mM Tris HCI 6 14 Place the AMPure bead solution back on magnet for 2 minutes 6 15 Carefully transfer 18 uL of the purified solution to a fresh 200 uL PCR tube or proceed directly to Step 7 Be sure to avoid transferring beads to the fresh tube 12 Archer Universal RNA Reagent Kit v2 for lon Torrent Platform 8 IFU AK0042 8 Rev C CH ARCHER a Stopping point It is OK to stop and store the library at 20 C Step 7 First PCR NOTE The Archer Universal RNA Reagent Kits
13. efly spin down and carefully remove remaining supernatant while taking care not to disturb the beads With caps open dry beads at room temperature for 5 minutes Take care not to over dry the beads as this can reduce yield 8 14 Remove tubes from the magnet and elute DNA by thoroughly resuspending the beads in 20 uL of 10 mM Tris HCI 8 15 Place the AMPure bead solution back on magnet for 2 minutes 8 16 Carefully transfer 18 uL of the purified solution to a fresh 200 uL PCR tube or proceed directly to Step 9 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 9 Quantify Library and Sequence 9 1 15 Use the KAPA Biosystems qPCR kit KK4827 for lon Torrent to quantify the concentration of each library Assume a 250 bp fragment length After quantification pool the barcoded libraries at equimolar concentrations and perform template preparation following the manufacturer 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 the number of samples per lon Torrent PGM 318 chip based on the coverage chart under Sample Multiplexing on page 5 of this manual Archer Universal RNA Reagent Kit v2 for lon Torrent Platfor
14. he tube 4 6 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 It is recommended to review the gPCR results from the Archer PreSeq RNA QC Assay Step 3 at this time to determine predicted sample success Step 5 End Repair dA Tailing 5 1 Gently open the End Repair dA Tailing SAQ004 foil pouch by tearing along the indents located at the top of the silver package 5 2 Remove the blue 8 tube strip Each tube in the strip provides a single reaction 5 2 1 If you would like to use fewer than 8 reactions carefully detach the appropriate number of tubes using clean scissors or a new razor blade Store the remaining unused tubes at 4 C in the sealed foil pouch with the provided desiccant 5 3 Centrifuge briefly to ensure lyophilized material is in the bottom of the tube 5 4 Transfer 40 uL of the Second Strand cDNA Synthesis reaction Step 4 6 into tube containing lyophilized End Repair dA Tailing SA0004 reagents After the lyophilized pellets dissolve mix well by gently pipetting up and down 6 8 times Spin briefly to collect contents at the bottom of the tube 5 5 Incubate the reaction in a thermal cycler with a heated lid set to gt 100 C and incubate as follows Step Incubation Temperature Incubation Time 1 12 C 15 min 2 37
15. iTect SYBR Green PCR Kits Cat 204141 lon SES Quality Control Kit Cat 4468656 ooo Er 10 lon 318 Chip Kit v2 Cat 4484355 11 lon PGM Template OT2 200 kit Cat 4480974 12 lon PGM Sequencing 200 kit v2 Cat 4482006 General Precautions Read the entire protocol before beginning Take note of stopping points where samples can be frozen at 20 C and plan your workflow accordingly Use good laboratory practices to minimize cross contamination of nucleic acid products Always use PCR tubes microfuge tubes and pipette tips that are certified sterile DNase and RNase free 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 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 Storage and Handling All components of the Archer Universal RNA Reagent Kit v2 Part AK0042 8 should be stored from 2 C to 8 C The liquid 10x VCP GSP1 and GSP2 primer tubes should be stored at 20 C Allow pouches to warm to room temperature before opening Archer Universal RNA Reagent Kit v2 for lon Torrent Platform 8 IFU AK0042 8 Rev C CH ARCHER a sample Multiplexing In order to efficiently utilize the throughput of the PGM multiple samples should be sequenced simultaneously Samples c
16. m 8 IFU AK0042 8 Rev C S ARCHER a 9 2 9 3 9 1 1 Samples within the pool should be demultiplexed 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 archerdx com mbc adapters 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 O12 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 may be adjusted accordingly to achieve the optimal percentage of unenriched template positive ISPs Upon completion of the run the data should be analyzed using Archer Analysis at http archerdx com analysis 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 2015 ArcherDX Inc All rights reserved Archer and Archer FusionPlex are trademarks of ArcherDX Inc lon Torrent P
17. m 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 Archer Universal RNA Reagent Kit v2 for lon Torrent Platform 8 IFU AK0042 8 Rev C CH ARCHER a Blat 3 min 1 95 C 30 sec 20 65 C ramp rate of 0 28 C sec 60 sec VIE 3 min 1 4 C HOLD 1 NOTE The percent of unique molecules will be reduced when the PCR cycles are increased The number of PCR cycles can be decreased based on user experience quantity of input material and specific sample types Post Second PCR AMPure XP Beads Purification 8 7 8 8 8 9 Add 24 uL of AMPure XP beads to the 20 uL reaction for a ratio of 1 2X Make sure that AMPure XP Beads are fully resuspended by vortexing prior to adding to the sample Vortex well or pipette 10 times to mix and visually inspect the color of the sample to ensure a homogenous mixture Incubate for 5 minutes at room temperature 8 10 Place beads on magnet for 2 4 minutes or until beads are pelleted and solution is clear 8 11 Carefully pipette off and discard supernatant without disturbing the beads 8 12 Without disturbing the separated beads wash by adding 200 uL of 70 ethanol while on the magnet and incubate for 30 sec at room temperature Carefully remove ethanol and discard Repeat this procedure for a total of 2 washes 8 13 After the final wash bri
18. on SA0022 4 Reagents a Step 1 Random Priming SA0001 x2 Step 2 First Strand cDNA Synthesis SA0002 x2 Step 3 PreSeq RNA QC Assay 10x VCP Primer Mix SA0121 Step 4 Second Strand cDNA Synthesis SA0003 x2 Step 5 End repair dA tailing SA0004 Step 6 Adapter Ligation SA0005 Step 7 First PCR SA0111 Step 8 Second PCR SA0112 Tom nm one 2 Archer Universal RNA Reagent Kit v2 for lon Torrent Platform 8 IFU AK0042 8 Rev C S ARCHER a Universal RNA Reagent Kit v2 Workflow Overview S ARCHER A Follow the instructions and incubations for each step All mixing steps should be performed on ice Pipette up and down 6 8 times to mix after re suspending each lyosphere and spin down prior to incubations and transfers For incubations use a lid heated to at least 5 degrees above the incuba tion temperature except where specified otherwise Add 20 250 ng RNA to Random Priming ineubation incubation EE Transfer 20 ul Random Priming mixture to First Strand cDNA Synthesis reagent step Ke Incubate as indicated Transfer 1 ul First Strand cDNA Synthesis incubation mixture to 9 ul water for PreSeq RNA QC Tempera Incubation of Assay Build qPCR reaction and incubate as ture Time cycles per your master mix specific instructions eee ft is These steps are specific TAREE for the iTaq Universal Kell eege SYBR Green Supermix DE Bio Rad amp 4 60 95 C increment Melt or default
19. rated into the Archer Universal RNA Reagent Kit v2 protocol between First Strand cDNA Synthesis and Second Strand cDNA Synthesis Steps 2 and 4 respectively A total of 16 reactions of Random Priming First Strand cDNA Synthesis and Second Strand cDNA Synthesis are included with the kit to help ensure that 8 high quality samples are identified for each batch of targeted library prep and sequencing Before You Begin e Make fresh 10 mM Tris HCl o Mix 20 ul 500 mM Tris HCl pH 8 0 SA0020 with 980 uL Ultra Pure Water SA0021 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 minimize evaporation 7 Archer Universal RNA Reagent Kit v2 for lon Torrent Platform 8 IFU AK0042 8 Rev C fm ARCHER we Protocol Step 1 Random Priming 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 1 3 1 If you would like to use fewer than 8 reactions carefully detach the appropriate number of tubes using clean scissors or a new razor blade Store the remaining unused tubes at 4 C in the seale
20. reat the extracted total nucleic acid with DNase as this will critically reduce the quality of RNA in the sample e If nucleic acid is from FFPE tissue it is recommended to use Agencourt FormaPure A33342 for extraction We do have modifications to the published instructions After digestion of FFPE samples at 55 C for 1 hour step 5 of the Agencourt protocol incubate at 80 C for 1 hour Additionally elute the sample in 40uL nuclease free water at step 23 This is the minimum elution volume 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 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 7 and 8 may need to be altered e As 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 HCI and not Tris EDTA buffer
21. t oes es ee ede vee ne ee ee ee neh nee 13 tee EE 14 Step 9 Quantify Library and Seouenceg 15 For more information please visit http www archerdn Com 16 1 Archer Universal RNA Reagent Kit v2 for lon Torrent Platform 8 IFU AK0042 8 Rev C ARCHER a Revision History From Rev To Rev Change Inclusion of Agencourt AMPure XP Bead purification in the End Repair dA Tailing step Altering the elution volumes related to AMPure XP Bead purification Updating workflow graphic to include Quick Start Guide Product Description Gene fusions represent an important class of genomic rearrangements in translational research The Archer Universal RNA Reagent Kit v2 and Archer 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 o s tu hybridization FISH Modular Assay Format The Archer Universal RNA Reagent Kit v2 for lon Torrent Platform used in conjunction with Archer FusionPlex Assays and Molecular Barcode MBC Adapters allows users to construct lon Torrent PGM ready libraries from total nucleic acid or RNA samples FusionPlex Assays Universal Reagent Kit Molecular Barcode MBC Adapters For Research Use Only Not for use in diagnostic procedures Kit Contents 1 500 mM Tris HCl pH 8 0 SA0020 2 Ultra Pure Water SA0021 3 Ultra Pure Water for Ethanol Diluti
22. tion Incubation Second PCR reagent Then mix and add 2 ul Temperature Time of cycles Saree 1 Incubate as indicated See 65 C panel specific cose Proton Optional stopping point 3 min 1 jj after this step Store at 20 C 4 Hold 1 AMPure XP clean up 1 2x Elute in 20 pl at Quantify normalize and sequence A Archer Universal RNA Reagent Kit v2 for lon Torrent Platform 8 IFU AK0042 8 Rev C fm ARCHER a Materials Required But Not Supplied NDAN 9 Archer MBC Adapters for lon Torrent Archer FusionPlex Assays or Custom Primer Panels designed at http assay archerdx com Agencourt AMPure XP Beads Cat A63881 Life Technologies DynaMag Cat 12331D 100 ethanol ACS grade KAPA Biosystems Library Quantification Kit lon Torrent Universal Cat KK4827 lf nucleic acid is from FFPE tissue it is recommended to use Agencourt FormaPure Cat A33342 for extraction SYBR qPCR Master Mix The Archer PreSeg RNA QC Assay has been validated for the use with the following commercially available SYBR master mixes If not using a master mix listed below please contact technical support at tech archerdx com a Recommended 2X BioRad iTaq Universal SYBR Green Super Mix Cat 172 5121 BioRad SsoAdvanced Universal SYBR Green Supermix Cat 172 5270 Life Technologies Power SYBR Green PCR Master Mix Cat 4368577 KAPA SYBR FAST GO Kits Cat KK4600 Qiagen Quant
23. tion JSC 20 sec 1 Denaturation 95 C 3 sec 35 Anneal Extension Data acquisition 60 C 30 sec Melt curve 60 C 95 C 0 5 C increment or default setting This protocol is for fast cycling conditions If your instrument requires standard cycling conditions increase the denaturation step and anneal extension step to 15 and 60 sec respectively NOTE gPCR can be run alongside Second Strand cDNA Synthesis Data generated can be reviewed during the stopping point after step 4 6 H Archer Universal RNA Reagent Kit v2 for lon Torrent Platform 8 IFU AK0042 8 Rev C S ARCHER a 4 1 Gently open the Second Strand cDNA Synthesis SA0003 foil pouch by tearing along the indents located at the top of the silver package 4 2 Remove the yellow 8 tube strip Each tube in the strip provides a single reaction 4 2 1 If you would like to use fewer than 8 reactions carefully detach the appropriate number of tubes carefully using clean scissors or a new razor blade Store the remaining unused tubes at 4 C in the sealed foil pouch with the provided desiccant 4 3 Centrifuge briefly to ensure lyophilized material is in the bottom of the tube 4 4 To the Second Strand cDNA Synthesis tube on ice add Ultra Pure Water SA0021 21 uL First Strand cDNA Synthesis reaction Step 2 6 19 uL Total 40 uL Ab Allow the pellets to dissolve and then gently pipet up and down 6 8 times to mix Spin briefly to collect contents at the bottom of t
24. tra Pure Water 5 14 Place the AMPure bead solution back on magnet for 2 minutes Adapter selection and addition 5 15 Gently open a pouch of Archer MBC Adapters for lon Torrent by tearing along the indents located at the top of the silver package 5 16 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 For example reactions 1 through 8 correspond to MBC Adapters 1 through 8 5 16 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 Track the index number added to each sample from this point forward 5162 If you would like to use fewer than 8 reactions carefully detach the appropriate number of tubes using clean scissors or a new razor blade Store the remaining unused tubes at 4 C in the sealed foil pouch with the provided desiccant Be sure to track which indices were used to ensure index compatibility when used in later experiments 5 17 Centrifuge briefly to ensure lyophilized material is in the bottom of the tube To the Archer MBC Adapters for lon Torrent tubes on ice add 50 uL of the purified End Repaired dA tailed DNA Step 5 14 5 18 Allow the pellet to dissolve and then gently pipet up and down 6 8 times to mix Spin briefly to collect contents at the bottom of the tube 5 19 Immediately proceed to
25. v2 do not contain gene specific primers GSPs in the reaction pellet Please use the Archer FusionPlex or custom assay GSPs 7 1 7 2 7 3 74 7 5 7 6 Gently open the First PCR SA0111 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 7 2 1 If you would like to use fewer than 8 reactions detach the appropriate number of tubes carefully using clean scissors or a new razor blade Store the remaining unused tubes at 4 C in the sealed foil pouch with the provided desiccant Centrifuge briefly to ensure lyophilized material is in the bottom of the tube To the First PCR tube on ice add Purified library DNA Step 6 15 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 95 C and 65 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 9540 3 min 1 95 C 30 sec 20 65 C ramp rate of 0 28 C sec 60 sec V2 3 min 1 4 C HOLD 1 NOTE The percent of unique molecules will be reduced when the PCR cycles are increase
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