SMARTer® Ultra Low Input RNA for Illumina® Sequencing
Contents
1. VI Covaris Shearing A Protocol Covaris Shearing of Full length cDNA Prior to generating the final library for Illumina sequencing the Covaris AFA system is used for controlled DNA shearing The resulting DNA will be in the 200 500 bp size range NOTE The full length cDNA output of the SMARTer Ultra Low Input RNA Kit for Illumina Sequencing can be processed using either the following protocol or our modified protocol for the Nextera DNA Sample Preparation kits from Illumina 1 Turn power ON for the Covaris system and the main cooler Add about 1 9 L of distilled or deionized water to the water bath The water level in the cooler should be within 3 mm of the FULL waterline when the transducer is submerged If needed add distilled or deionized water to the water bath until the FULL line is reached Important Never run a process without the water bath This will permanently damage the transducer 2 Close the door and open the Sonolab software Click ON for the degassed button and degas the water bath for 1 2 hour 30 minutes 3 Add 65 ul of Purification Buffer to the DNA from Section V A Step 12 Transfer 75 ul of the Purification Buffer DNA mixture into the 100 ul Covaris tube Put the sample tubes into the appropriate location on the Sample holder 4 Set up the process configuration panel based on the following table Table 3 Process Configuration Panel Setup Duty Intensity Bu2. 072313 www clontech com Page 12 of 17 Clontech Laboratories Inc A Takara Bio Company SMARTer Ultra Low Input RNA for Illumina Sequencing HV User Manual Protocol ds cDNA Amplification by LD PCR Perform Steps 1 amp 2 in PCR Clean Work Station We strongly recommend using the Advantage 2 PCR Kit included in this kit also sold separately as Cat Nos 639206 amp 639207 for PCR amplification The Advantage 2 Polymerase Mix has been specially formulated for efficient and accurate amplification of cDNA templates by long distance PCR LD PCR Barnes 1994 Table 2 provides guidelines for optimizing your PCR depending on the amount of total RNA used in the first strand synthesis IMPORTANT Optimal parameters may vary for different templates and thermal cyclers To determine the optimal number of cycles for your sample and conditions we strongly recommend that you perform a range of cycles Do not exceed the recommended cycle numbers in the table below for different starting amounts of material Table 2 Cycling Guidelines Based on Amount of Starting Material Input Amount Input Amount Typical No of Total RNA Cells PCR Cycles 10 ng 1 000 cells 12 1 ng 100 cells 12 500 pg 50 cells 13 100 pg 10 cells 15 10 pg 1 cell 18 1 Prepare enough PCR Master Mix for all the reactions plus 10 of the total reaction mix volume Combine the following reagents in the order shown then mix well by vortexin
3. eene nren eren nenn tenente nnne 13 V Amplified cDNA Purification amp Validation cente ete bete tee eee err due Edge tne koe eaae rata dvo iea 14 A Protocol Purification of ds cDNA using SPRI Ampure Beads eese 14 B Validation Using the Agilent 2100 BioAnalyzer eese rennen nnne nenn tenete teen nne 15 BASE SENI 16 A Protocol Covaris Shearing of Full length cDNA esses nennen nne nennen nennen tnnt nnne 16 MIHI X 16 Table of Figures Figure 1 Protocol Overviews P 3 Figure 2 Flowchart of SMARTer cDNA synthesis esee nennen tenete nnne en rennen eren 4 Figure 3 Optional setup to ensure proper and steady positioning of tubes containing first strand cDNA 12 Figure 4 Electropherogram example results from Agilent 2100 Bioanalyzer sese 15 Table of Tables Table 1 Sample Preparation Guidelines eese en rennen e EN a E a aS treten 11 Table 2 Cycling Guidelines Based on Amount of Starting Material sese nennen 13 Table 3 Process Configuration Panel Setup sessi enne een rennen nne ne st nes ntes ennt tnne tenente tnnt 16 072313 www clontech com Page 2 of 17 Clontech Laboratories Inc A Takara Bio
4. Add the reverse transcriptase to the master mix just prior to use Mix well by gently vortexing and spin the tube s briefly in a microcentrifuge NOTE The SMARTer Ultra Low Input RNA for Illumina Sequencing Components HV Cat Nos 634822 634825 6348277 amp 634831 and the Advantage 2 PCR Kit Cat Nos 639206 amp 639207 both include dNTP mixes Use the SMARTer dNTP Mix 20 mM each dNTP for first strand cDNA synthesis 6 Add 9 ul of the Master Mix to each reaction tube from Step 4 Mix the contents of the tubes by gently pipetting and spin them briefly to collect the contents at the bottom 7 Place the tubes in a preheated thermocycler and incubate at 42 C for 90 min 8 Terminate the reaction by heating the tube s at 70 C for 10 min NOTE The tubes can be stored at 4 C overnight 072313 www clontech com Page 11 of 17 Clontech Laboratories Inc A Takara Bio Company SMARTer Ultra Low Input RNA for Illumina Sequencing HV User Manual F Protocol Purification of First Strand cDNA using SPRI Ampure Beads Perform in PCR Clean Work Station The first strand cDNA selectively binds to SPRI beads leaving contaminants in solution which is removed by a magnetic separation The beads are then directly used for PCR amplification NOTES e Aliquot SPRI beads prior to use e Before use bring beads to room temperature and mix well to disperse e In order to ensure proper and steady positioning of the tubes containing fi
5. instructions 2 Compare the results for your samples amp controls see Figure 4 to verify whether the sample is suitable for further processing Successful cDNA synthesis and amplification should yield no product in the negative control Figure 4 Panel B and a distinct peak spanning 400 bp to 9 000 bp peaked at 2 000 bp for the positive control RNA sample Figure 4 Panel A yielding approximately 2 7 ng of cDNA depending on the input Contaminated samples will have a broader peak and an abnormally high yield Figure 4 Panel C 3 Proceed to Section VI Covaris Shearing Positive Control RNA Negative Control Fu 100pg 15cyc nc 18 TT TT toT 35 100 200 300 400 500 700 2000 10380 bp 35 100 200 300 400 600 1000 3000 10380 bp Contaminated Sample FU 4 sample 3 35 100 200 300 400 600 1000 3000 10380 bp Figure 4 Electropherogram example results from Agilent 2100 Bioanalyzer All samples were subjected to SMARTer cDNA synthesis and amplification as described in the protocol FU fluorescence absorption units Panel A top left Clean SMARTer Amplification Product 15 PCR cycles Panel B top right Clean SMART Negative Control 18 PCR cycles Panel C bottom Example of Contaminated SMARTer Amplification Product 072313 www clontech com Page 15 of 17 Clontech Laboratories Inc A Takara Bio Company SMARTer Ultra Low Input RNA for Illumina Sequencing HV User Manual
6. 0 ul 200 ul 200 ul 50 ul 30 ul 30 ul 2x 1 25 ml 50X Advantage 2 Polymerase Mix 10X Advantage 2 PCR Buffer 10X Advantage 2 SA PCR Buffer 50X dNTP Mix 10 mM each Control DNA Template 100 ng ul Control Primer Mix 10 uM each PCR Grade Water SMARTer Ultra Low Input RNA for Illumina Sequencing HV 48 rxns Cat No 634826 e 48rxns The SMARTer Ultra Low Input RNA for Illumina Sequencing Components HV Cat No 634827 Not sold separately Box 1 48 ul 5 ul Box 2 48 ul 96 ul 192 ul 48 ul 48 ul 96 ul 5 ml 264 ul 5 ml 5 ml SMARTer II A Oligonucleotide 24 uM Control Total RNA 1 ug l 3 SMART CDS Primer II A 24 uM IS PCR Primer 12 uM 5X First Strand Buffer RNase Free SMARTer dNTP Mix dATP dCTP dGTP and dTTP each at 20 mM Dithiothreitol DTT 100 mM SMARTScribe Reverse Transcriptase 100 U l Nuclease Free Water RNase Inhibitor 40 U ul Dilution Buffer Purification Buffer 10 mM Tris Cl pH 8 5 e 100 rxns Advantage 2 PCR Kit Cat No 639206 100 ul 600 ul 600 ul 120 ul 100 ul 100 ul 4x 1 25 ml 50X Advantage 2 Polymerase Mix 10X Advantage 2 PCR Buffer 10X Advantage 2 SA PCR Buffer 50X dNTP Mix 10 mM each Control DNA Template 100 ng ul Control Primer Mix 10 uM each PCR Grade Water 072313 www clontech com Clontech Laboratories Inc A Takara Bio Company Page 6 of 17 SMARTer Ultra Low Input RNA for Illumina Sequencing HV User Manual SMARTer Ul
7. Clontech Laboratories Inc SMARTer Ultra Low Input RNA for Illumina sequencing HV User Manual Cat Nos 634820 634823 634826 634828 amp 634830 072313 Clontech Laboratories Inc A Takara Bio Company 1290 Terra Bella Avenue Mountain View CA 94043 USA U S Technical Support tech clontech com United States Canada Asia Pacific Europe Japan 800 662 2566 1 650 919 7300 33 0 1 3904 6880 amp 81 0 77 543 6116 Page 1 of 17 SMARTer Ultra Low Input RNA for Illumina Sequencing HV User Manual Table of Contents IM ore iei M EEE E 3 I So dero nmoSUMte E M 5 III Additional Materials Required esses nnne nnne nnne tnnetn netten enr et eren nest rese nesn nennen nne 8 IEEE NES Su CDNA EIU EE 9 A Requirements for Preventing Contamination eese sees ener entren enr en eren nr enne senes nennen 9 lO SnoriBiGe rns 9 C Sample RecomimenGatOns ER m 10 D Sample Requirements cec Lp eiie tei iet d tei OR RED een ee LO aes 10 E Protocol First Strand cDNA Synthesis esses eene ener nnne ener enr en eren nenne 10 F Protocol Purification of First Strand cDNA using SPRI Ampure Beads esee 12 G Protocol ds cDNA Amplification by LD PCR
8. Company SMARTer Ultra Low Input RNA for Illumina Sequencing HV User Manual l Introduction SMARTer cDNA Synthesis for the Illumina Sequencing Platform The SMARTer Ultra Low Input RNA for Illumina Sequencing HV kits Cat Nos 634820 634823 634826 634828 amp 634830 allow high quality cDNA synthesis starting from as little as 10 pg of total RNA or cells in an input volume of up to 9 ul Our original SMARTer Ultra Low Input RNA Kit for Ilumina Sequencing accommodates input volumes of up to 1 pl The kits have been designed and validated to prepare cDNA samples for sequencing and quantitation with the Illumina HiSeq amp MiSeq and Genome Analyzer sequencing instruments The entire library construction protocol can be completed within two days Figure 1 SMART technology offers unparalleled sensitivity and unbiased amplification of cDNA transcripts enabling a direct start from your sample Most importantly SMART technology enriches for full length transcripts and maintains the true representation of the original mRNA transcripts these factors are critical for transcriptome sequencing and gene expression analysis Start with Total RNA or Cells Section IV C SMARTer First strand cDNA Synthesis amp Purification Sections IV E amp IV F Full Length ds cDNA Amplification by LD PCR Section IV G ouo Aeg Amplified cDNA Purification amp Valdation Sections V A amp V B Covaris Shearing of Full Length cD
9. Inc Clontech the Clontech logo Advantage SMART SMARTer SMARTScribe and Ultra are trademarks of Clontech Laboratories Inc All other marks are the property of their respective owners Certain trademarks may not be registered in all jurisdictions Clontech is a Takara Bio Company 2013 Clontech Laboratories Inc This document has been reviewed and approved by the Clontech Quality Assurance Department 072313 www clontech com Page 17 of 17 Clontech Laboratories Inc A Takara Bio Company
10. NA Section VI Library preparation i Using the Low Input Library Prep Kit Cat No 634947 E lt z Cluster Generation Figure 1 Protocol Overview 072313 www clontech com Page 3 of 17 Clontech Laboratories Inc A Takara Bio Company SMARTer Ultra Low Input RNA for Illumina Sequencing HV User Manual SMARTer cDNA synthesis starts with picogram amounts of total RNA A modified oligo dT primer the SMART CDS Primer primes the first strand synthesis reaction Figure 2 When SMARTScribe Reverse Transcriptase reaches the 5 end of the mRNA the enzyme s terminal transferase activity adds a few additional nucleotides to the 3 end of the cDNA The carefully designed SMARTer Oligonucleotide base pairs with the non template nucleotide stretch creating an extended template to enable SMARTScribe RT continue replicating to the end of the oligonucleotide Chenchik et al 1998 The resulting full length single stranded ss cDNA contains the complete 5 end of the mRNA as well as sequences that are complementary to the SMARTer Oligonucleotide In cases where the RT pauses before the end of the template the addition of non template nucleotides is much less efficient than with full length cDNA RNA hybrids thus the overhang needed for base pairing with the SMARTer Oligonucleotide is absent The SMARTer anchor sequence and the poly A sequence serve as universal priming sites for end to end cDNA amplification In contrast
11. Sample 1 9 ul Total Volume 10 ul 10 ul 10 ul The Control RNA is supplied at a concentration of 1 ug ul The Control RNA should be diluted in nuclease free water to match the concentration of your test sample Perform serial dilutions on the Control RNA until you obtain the appropriate concentration Place the samples on a 20 C prechilled IsoFreeze PCR rack in a PCR clean station and add 1 ul of 3 SMART CDS Primer II A 24 uM Mix the contents and spin the tube s briefly in a microcentrifuge 10 ul Cell Total RNA in Reaction Buffer from Table 1 1gl 3 SMART CDS Primer Il A 24 uM 11 pl Total Volume Place the tubes into a preheated thermocycler and incubate the tube s at 72 C in a hot lid thermal cycler for 3 min then put the samples on the IsoFreeze PCR rack NOTE The initial reaction steps Step 6 8 are critical for first strand synthesis and should not be delayed after completing Step 4 You can prepare your master mix for Step 5 while your tubes are incubating Step 4 in order to jump start the cDNA synthesis Meanwhile prepare enough Master Mix for all the reactions plus 1046 of the total reaction mix volume by combining the following reagents in the order shown at room temperature 4ul 5X First Strand Buffer 0 5 ul DTT 100 mM 1 ul dNTP Mix 20 mM 1 ul SMARTer IIA Oligonucleotide 24 uM 0 5 ul RNase Inhibitor 2ul SMARTScribe Reverse Transcriptase 100 U 9pl Total Volume added per reaction
12. User Manual IV SMARTer cDNA Synthesis NOTE Please read the entire protocol before starting This protocol is optimized for the generation of cDNA starting from ultra low amounts of total RNA using Clontech s SMART technology The protocol also works starting from cells Due to the sensitivity of the protocol the input material total RNA or cells needs to be collected and purified under clean room conditions to avoid contamination The whole process of SMARTer cDNA Synthesis should be carried out in a PCR Clean Work Station under clean room conditions A Requirements for Preventing Contamination Before you set up the experiment make sure you have two physically separated work stations A PCR Clean Work Station for all pre PCR experiments that require clean room conditions e g first strand cDNA synthesis Protocol IV E and first strand cDNA purification Protocol IV F NOTES The PCR Clean Work Station must be located in a clean room with positive air flow as contamination may occur very easily Once contamination occurs it can be difficult to remove Strictly obey clean room operation rules A second work station located in the general laboratory where you will perform PCR Protocol IV G and measure cDNA concentration Protocol V B B General Requirements The success of your experiment depends on the quality of your starting sample of RNA Prior to cDNA synthesis please make sure that your RNA is intact and free o
13. and dTTP each at 20 mM Dithiothreitol DTT 100 mM SMARTScribe Reverse Transcriptase 100 U l Nuclease Free Water RNase Inhibitor 40 U ul Dilution Buffer Purification Buffer 10 mM Tris Cl pH 8 5 30 rxns Advantage 2 PCR Kit Cat No 639207 30 ul 200 ul 200 ul 50 ul 30 ul 30 ul 2x1 25ml 50X Advantage 2 Polymerase Mix 10X Advantage 2 PCR Buffer 10X Advantage 2 SA PCR Buffer 50X dNTP Mix 10 mM each Control DNA Template 100 ng ul Control Primer Mix 10 uM each PCR Grade Water 072313 www clontech com Clontech Laboratories Inc A Takara Bio Company Page 5 of 17 SMARTer Ultra Low Input RNA for Illumina Sequencing HV User Manual SMARTer Ultra Low Input RNA for Illumina Sequencing HV 24 rxns Cat No 634823 e 24rxns The SMARTer Ultra Low Input RNA for Illumina Sequencing Components HV Cat No 634825 Not sold separately Box 1 24 ul 5 ul Box 2 24 ul 48 ul 96 ul 24 ul 24 ul 48 ul 2 ml 132 ul 2 ml 2 ml SMARTer II A Oligonucleotide 24 uM Control Total RNA 1 ug l 3 SMART CDS Primer II A 24 uM IS PCR Primer 12 uM 5X First Strand Buffer RNase Free SMARTer dNTP Mix dATP dCTP dGTP and dTTP each at 20 mM Dithiothreitol DTT 100 mM SMARTScribe Reverse Transcriptase 100 U ul Nuclease Free Water RNase Inhibitor 40 U ul Dilution Buffer Purification Buffer 10 mM Tris Cl pH 8 5 e 2x 30rxns Advantage 2 PCR Kit Cat No 639207 3
14. cDNA without these sequences such as prematurely terminated cDNAs contaminating genomic DNA or cDNA transcribed from poly A RNA will not be exponentially amplified However truncated RNAs with poly A tails that are present in poor quality RNA starting material will be amplified yielding shorter cDNA fragments Total RNA or cell s yxX BEND DSSS IIS SSS poly A 3 X t SMART CDS primer SMARTer Il A Oligonucleotide First strand synthesis and tailing by RT l DECA BE DPSS SPP yox Template switching and extension by RT 5 blocked primer X ooo Avava AVAVA VAVA VATA TATATATA ATA An 5 yoQ ok Amplify cDNA by LD PCR with IS PCR primer Double stranded cDNA dec is n 5r Figure 2 Flowchart of SMARTer cDNA synthesis The SMARTer II A Oligonucleotide 3 SMART CDS Primer II A and IS PCR Primer all contain a stretch of identical sequence 072313 www clontech com Page 4 of 17 Clontech Laboratories Inc A Takara Bio Company List of Components SMARTer Ultra Low Input RNA for Illumina Sequencing HV User Manual The following components have been specifically designed to work together and are optimized for this particular protocol Please do not make any substitutions The substitution of reagents in the kit and or a modification of the protocol may lead to unexpected results The SMARTer Ultra Low Input RNA for Illumina Sequencing HV kits Cat Nos 634820 634823 634826 634828 amp 634830 consist of e T
15. f contaminants The assay is very sensitive to variations in pipette volume etc Please make sure all your pipettes are calibrated for reliable delivery and make sure nothing is attached to the outside of the tips All lab supplies related to SMARTer cDNA synthesis need to be stored in a DNA free closed cabinet Reagents for SMARTer cDNA synthesis need to be stored in a freezer refrigerator that has not previously been used to store PCR amplicons Add enzymes to reaction mixtures last and thoroughly incorporate them by gently pipetting the reaction mixture up and down Do not increase or decrease the amount of enzyme added or the concentration of DNA in the reactions The amounts and concentrations have been carefully optimized for the SMARTer amplification reagents and protocol If you are using this protocol for the first time we strongly recommend that you perform negative and positive control reactions to verify that kit components are working properly 072313 www clontech com Page 9 of 17 Clontech Laboratories Inc A Takara Bio Company SMARTer Ultra Low Input RNA for Illumina Sequencing HV User Manual Sample Recommendations The sequence complexity and the average length of SMARTer cDNA are noticeably dependent on the quality of starting RNA material Due to the limiting sample size most traditional RNA isolation methods may not be applicable There are several commercially available products that enable purification of to
16. fication Buffer 1 Take out a 96 well Axygen V bottom plate and cover all the wells with a MicroAmp Clean Adhesive Seal Uncover only the wells that you want to use Vortex SPRI beads till even and then add 90 ul of SPRI Ampure XP Beads to the wells of the 96 well plate 2 Transfer the entire PCR product including the SPRI beads from Section IV G Step 3 to the wells of the plate containing the SPRI beads from Step 1 above Pipette the entire volume up and down 10 times to mix thoroughly Incubate at room temperature for 8 min to let the DNA bind to the beads NOTE The beads are viscous suck the entire volume up and push it out slowly 3 Place the 96 well plate on the Ambion Magnetic Stand 96 for 5 min or longer until the liquid appears completely clear and there are no beads left in the supernatant 4 While the plate is sitting on the magnetic stand pipette out the supernatant 5 Keep the plate on the magnetic stand Add 200 ul of freshly made 80 Ethanol to each sample without disturbing the beads to wash away contaminants Wait for 30 seconds and carefully pipette out the supernatant DNA will remain bound to the beads during the washing process 6 Repeat Step 5 one more time 7 Sealthe sample wells on the plate and briefly spin down for 10 seconds at 1 000 rpm to collect the liquid at the bottom of the well 8 Place the 96 well plate on the magnetic stand for 30 seconds then remove all the remaining Ethanol 9 Place the pla
17. g and spin the tube briefly in a microcentrifuge 5byl 10X Advantage 2 PCR Buffer 2ul dNTP Mix 10 mM 2ul IS PCR Primer 12 uM 2ul 50X Advantage 2 Polymerase Mix 39 ul Nuclease Free Water 50 ul Total Volume per reaction NOTE The SMARTer Ultra Low Input RNA for Illumina Sequencing Components HV Cat Nos 634822 634825 634827 amp 634831 and the Advantage 2 PCR Kit Cat Nos 639206 amp 639207 both include dNTP mixes Use the Advantage 2 dNTP Mix 10 mM each dNTP for ds cDNA amplification 2 Add 50 ul of PCR Master Mix to each tube containing DNA bound to the beads from Section IV F Step 4 Mix well and briefly spin down Important Transfer the samples from the PCR Clean Work Station to the general lab All downstream processes will be performed in the general lab 3 Place the tube in a preheated thermal cycler with a heated lid Commence thermal cycling using the following program 95 C 1 min X cycles o 95 C 15sec 65 30sec 68 C 6min 72 C 10 min 4 C forever Consult Table 2 for guidelines 072313 www clontech com Page 13 of 17 Clontech Laboratories Inc A Takara Bio Company SMARTer Ultra Low Input RNA for Illumina Sequencing HV User Manual V Amplified cDNA Purification amp Validation A Protocol Purification of ds cDNA using SPRI Ampure Beads PCR amplified cDNA is purified by immobilizing it onto SPRI beads The beads are then washed with 80 Ethanol and eluted in Puri
18. he Advantage 2 PCR Kit Cat No 639206 or 639207 which has been specially formulated to provide automatic hot start PCR Kellogg et al 1994 and can efficiently amplify full length cDNAs with a significantly lower error rate than that of conventional PCR Barnes 1994 e The SMARTer Ultra Low RNA Kit for Illumina Sequencing Components Cat No 634822 634825 634827 or 634831 NOTES e The SMARTer Ultra Low Input RNA for Illumina Sequencing Components HV Cat Nos 634822 634825 634827 amp 634831 and the Advantage 2 PCR Kit Cat Nos 639206 amp 639207 both include dNTP mixes Use the SMARTer dNTP Mix 20 mM each dNTP for first strand cDNA synthesis Step IV E 5 Use the Advantage 2 dNTP Mix 10 mM each dNTP for double stranded cDNA amplification Step IV G 1 e Donotuse the Advantage 2 SA Buffer supplied with the Advantage 2 PCR Kit with this SMARTer cDNA synthesis protocol The specific composition of each kit is as follows SMARTer Ultra Low Input RNA for Illumina Sequencing HV 12 rxns Cat No 634820 12 rxns SMARTer Ultra Low Input RNA for Illumina Sequencing Components HV Cat No 634822 Not sold separately Box 1 12 ul 5 ul Box 2 12 ul 24 ul 48 ul 12 ul 12 ul 24 ul 1 ml 66 ul 1 ml 1 ml SMARTer II A Oligonucleotide 24 uM Control Total RNA 1 yg pl 3 SMART CDS Primer Il A 24 uM IS PCR Primer 12 uM 5X First Strand Buffer RNase Free SMARTer dNTP Mix dATP dCTP dGTP
19. l RNA 1 ug l 3 SMART CDS Primer II A 24 uM IS PCR Primer 12 uM 5X First Strand Buffer RNase Free SMARTer dNTP Mix dATP dCTP dGTP and dTTP each at 20 mM Dithiothreitol DTT 100 mM SMARTScribe Reverse Transcriptase 100 U ul Nuclease Free Water RNase Inhibitor 40 U ul Dilution Buffer Purification Buffer 10 mM Tris Cl pH 8 5 e 10x 100 rxns Advantage 2 PCR Kit Cat No 639206 100 ul 600 ul 600 ul 120 ul 100 ul 100 ul 4x 1 25 ml 50X Advantage 2 Polymerase Mix 10X Advantage 2 PCR Buffer 10X Advantage 2 SA PCR Buffer 50X dNTP Mix 10 mM each Control DNA Template 100 ng ul Control Primer Mix 10 uM each PCR Grade Water 072313 www clontech com Clontech Laboratories Inc A Takara Bio Company Page 7 of 17 SMARTer Ultra Low Input RNA for Illumina Sequencing HV User Manual Storage Conditions e Store Control Total RNA and SMARTer IIA Oligonucleotide at 70 C e Store Dilution Buffer at 20 C Once thawed the buffer can be stored at 4 C e Store Purification Buffer at 20 C Once thawed the buffer can be stored at Room Temperature e Store all other reagents at 20 C Ill Additional Materials Required The following reagents are required but not supplied These materials have been validated to work with this protocol Please do not make any substitutions because you may not obtain the expected results Single channel pipette 10 ul 20 ul and 200 ul one each Eight cha
20. lean Work Station IMPORTANT To avoid introducing contaminants into your RNA sample the first part of the cDNA synthesis protocol Sections E G requires use of a PCR work station in a clean room Standard clean room procedure should be followed If no clean room is available you may work with just a PCR Clean Work Station on a temporary basis We strongly recommend putting the PCR work station in a clean room to avoid contamination It is critical to have an air blower in the PCR work station turned on during the whole process 1 Prepare a stock solution of Reaction Buffer by mixing the Dilution Buffer with the RNase Inhibitor as indicated below scale up as needed 19 ul Dilution Buffer 1 ul RNase Inhibitor 20 ul Total Volume 2 See Table 1 for guidelines on setting up your control and test samples Prepare each sample 10 ul total volume in individual 0 2 ml RNase free PCR tubes in an 8 well strip Add 1 5 ul of Reaction Buffer and 1 9 ul of cells or RNA Use the same volume of Reaction Buffer in the negative and positive controls as in your sample 072313 www clontech com Page 10 of 17 Clontech Laboratories Inc A Takara Bio Company SMARTer Ultra Low Input RNA for Illumina Sequencing HV User Manual Table 1 Sample Preparation Guidelines Components Negative Control Positive Control Test Sample Reaction Buffer 1 5 ul 1 5 ul 1 5 ul Nuclease free water 1 9 ul 0 4 ul 0 4 ul Diluted Control RNA 1 9 ul
21. nnel pipette 20 ul and 200 ul one each Filter pipette tips 10 ul 20 ul and 200 ul one box each One QuickSpin minicentrifuge for 1 5 ml tubes One QuickSpin minicentrifuge for 0 2 ml tubes For PCR Amplification amp Validation One dedicated PCR thermal cycler used only for first strand synthesis High Sensitivity DNA Kit Agilent Cat No 5067 4626 IsoFreeze Flipper Rack MIDSCI Cat No TFL 20 IsoFreeze PCR Rack MIDSCI Cat No 5640 T4 Nuclease free thin wall PCR tubes 0 2 ml USA Scientific Cat No 1402 4700 Nuclease free nonsticky 1 5 ml tubes USA Scientific Cat No 1415 2600 For SPRI Bead Purification Agencourt AMPure PCR Purification Kit 5 ml Beckman Coulter Part No A63880 60 ml Beckman Coulter Part No A63881 Use this kit for the SPRI Purifications Sections IV F amp V A MagnaBot II Magnetic Separation Device Promega Part No V8351 Use this stand for the first purification Section IV F Magnetic Stand 96 Ambion Part No AM10027 Use this stand for the second purification Section V A 96 well V bottom Plate 500 ul VWR Cat No 47743 996 MicroAmp Clean Adhesive Seal AB Part No 4306311 8096 Ethanol For Sequencing Library Generation Low Input Library Prep Kit Cat No 634947 Covaris Instrument and Related Materials for DNA Shearing 072313 www clontech com Page 8 of 17 Clontech Laboratories Inc A Takara Bio Company SMARTer Ultra Low Input RNA for Illumina Sequencing HV
22. ontact Us For Assistance Customer Service Ordering Technical Support Telephone 800 662 2566 toll free Telephone 800 662 2566 toll free Fax 800 424 1350 toll free Fax 800 424 1350 toll free Web www clontech com Web www clontech com E mail orders clontech com E mail tech clontech com Notice to Purchaser Hw The Low Input Library Prep Kit contains ThruPLEX FD technology developed and manufactured by Rubicon Genomics Inc Ann Arbor RUBICON GENOMICS Michigan USA and protected by US Patent 7 803 550 EP1924704 and US and international patents pending Clontech products are to be used for research purposes only They may not be used for any other purpose including but not limited to use in drugs in vitro diagnostic purposes therapeutics or in humans Clontech products may not be transferred to third parties resold modified for resale or used to manufacture commercial products or to provide a service to third parties without prior written approval of Clontech Laboratories Inc Your use of this product is also subject to compliance with any applicable licensing requirements described on the product s web page at http www clontech com It is your responsibility to review understand and adhere to any restrictions imposed by such statements Illumina Genome Analyzer HiSeq MiSeq and Nextera are registered trademarks or trademarks of Illumina Inc ThruPLEX FD is a trademark of Rubicon Genomics
23. rst Cycle Time min Mode 10 5 200 5 min Frequency Sweeping 5 Save the file and click return to go back to the main page 6 Open the door Place the tube holder with sample tubes on the transducer positioning system 7 Close the door 8 Click START on the main page to run the process 9 After shearing is complete transfer 75 ul of sheared DNA to 1 5 ml tubes 10 Proceed to generate an Illumina Sequencing Library with the Low Input Library Prep Kit Cat No 634947 Dispose all tubes and pipettes that have been exposed to amplicons in a sealed trash bag Vil References Barnes W M 1994 PCR amplification of up to 35 kb DNA with high fidelity and high yield from X bacteriophage templates Proc Natl Acad Sci USA 91 2216 2220 Chenchik A Zhu Y Diatchenko L Li R Hill J amp Siebert P 1998 Generation and use of high quality cDNA from small amounts of total RNA by SMART PCR In RT PCR Methods for Gene Cloning and Analysis Eds Siebert P amp Larrick J BioTechniques Books MA pp 305 319 Kellogg D E Rybalkin I Chen S Mukhamedova N Vlasik T Siebert P amp Chenchik A 1994 TaqStart Antibody Hot start PCR facilitated by a neutralizing monoclonal antibody directed against Tag DNA polymerase BioTechniques 16 1134 1137 072313 www clontech com Page 16 of 17 Clontech Laboratories Inc A Takara Bio Company SMARTer Ultra Low Input RNA for Illumina Sequencing HV User Manual C
24. rst strand cDNA from Protocol E you may place the tubes in the top part of an inverted P20 or P200 Rainin Tip Holder which is taped to the MagnaBlot II Magnetic Separator Figure 3 Optional setup to ensure proper and steady positioning of tubes containing first strand cDNA To purify the SMART cDNA from unincorporated nucleotides and small 0 1 kb cDNA fragments follow this procedure for each reaction tube 1 Add 36 ul of SPRI Ampure XP beads to each sample using a 200 ul pipetter Adjust the pipetter to 56 ul and pipette the entire volume up and down 10 times to mix thoroughly The beads are viscous suck the entire volume up and push it out slowly Incubate at room temperature for 8 minutes to let DNA bind to the beads 2 Briefly spin the sample tubes to collect the liquid from the side of the wall Place the sample tubes on the Promega MagnaBot II Magnetic Separation Device for 5 min or longer until the solution is completely clear 3 While samples are still on the Magnetic Separation Device pipette out the solution and discard Briefly spin the tubes to collect the liquid at the bottom 4 Place the tubes back in the Promega MagnaBot II Magnetic Separation Device for 2 min or longer to let beads separate from the liquid completely Pipette out the residual liquid from the beads using a 10 ul pipetter and discard Make sure that there is no supernatant remaining in the tube Be careful not to remove any beads with the supernatant
25. tal RNA preparations from extremely small samples e g Clontech offers the NucleoSpin RNA XS Kit Cat No 740902 10 for purification of RNA from 10 cells When choosing a purification method kit ensure that it is appropriate for your sample amount e Although SMART Technology is sensitive enough to generate cDNA from as little as 10 pg of total RNA the use of a higher amount of starting material 100 pg to 10 ng is recommended for reproducible amplification of low abundance mRNA transcripts e After RNA extraction if your sample size is not limiting we recommend evaluating total RNA quality using the Agilent RNA 6000 Pico Kit Cat No 5067 1513 Sample Requirements The original SMARTer Ultra Low RNA Kit for lumina Sequencing Cat No 634936 works with a 1 ul sample containing your cells or RNA We have optimized SMARTer Ultra Low Input RNA for Illumina Sequencing HV Cat Nos 634820 634823 634826 634828 and 634830 to work with up to 9 ul of cells or RNA e Total RNA This protocol has been optimized for cDNA synthesis starting from 10 pg of total RNA However if your RNA sample is not limiting we recommend that you start with more total RNA up to 10 ng Purified total RNA should be in nuclease free water e Cells Although this protocol was optimized for cDNA synthesis starting from total RNA the protocol has also been validated to work starting from cells Protocol First Strand cDNA Synthesis Perform in PCR C
26. te at room temperature for 3 5 min until the pellet appears dry You may see a tiny crack in the pellet when it is dry NOTE Be sure to dry the pellet enough If you under dry the pellet ethanol will remain in the sample wells The ethanol will reduce your ds cDNA recovery rate and ultimately your yield Allow the plate to sit at room temperature until the pellet is dry If you over dry the pellet it will take longer than 2 min to rehydrate Step V A 10 10 Once the beads are dried add 12 ul of Purification Buffer to cover the beads Remove the plate from the magnetic stand and incubate at room temperature for 2 min to rehydrate 11 Mix the pellet by pipetting up and down 10 times to elute DNA from the beads then put the plate back on the magnetic stand for 1 minute or longer until the solution is completely clear 12 Transfer clear supernatant containing purified cDNA from each well to a nuclease free nonsticky tube Label each tube with sample information and store at 20 C 072313 www clontech com Page 14 of 17 Clontech Laboratories Inc A Takara Bio Company SMARTer Ultra Low Input RNA for Illumina Sequencing HV User Manual B Validation Using the Agilent 2100 BioAnalyzer 1 Aliquot 1 ul of the amplified cDNA for validation using the Agilent 2100 BioAnalyzer and the High Sensitivity DNA Chip from Agilent s High Sensitivity DNA Kit Cat No 5067 4626 See the user manual for the Agilent High Sensitivity DNA Kit for
27. tra Low Input RNA for Illumina Sequencing HV 96 rxns Cat No 634828 e 96rxns The SMARTer Ultra Low Input RNA for Illumina Sequencing Components HV Cat No 634831 Not sold separately Box 1 96 ul 5 ul Box 2 96 ul 192 ul 384 ul 96 ul 96 ul 192 ul 10 ml 528 ul 10 ml 10 ml SMARTer II A Oligonucleotide 24 uM Control Total RNA 1 ug l 3 SMART CDS Primer II A 24 uM IS PCR Primer 12 uM 5X First Strand Buffer RNase Free SMARTer dNTP Mix dATP dCTP dGTP and dTTP each at 20 mM Dithiothreitol DTT 100 mM SMARTScribe Reverse Transcriptase 100 U l Nuclease Free Water RNase Inhibitor 40 U ul Dilution Buffer Purification Buffer 10 mM Tris Cl pH 8 5 e 2x 100 rxns Advantage 2 PCR Kit Cat No 639206 100 ul 600 ul 600 ul 120 ul 100 ul 100 ul 4x 1 25 ml 50X Advantage 2 Polymerase Mix 10X Advantage 2 PCR Buffer 10X Advantage 2 SA PCR Buffer 50X dNTP Mix 10 mM each Control DNA Template 100 ng ul Control Primer Mix 10 uM each PCR Grade Water SMARTer Ultra Low Input RNA for Illumina Sequencing HV 480 rxns Cat No 634830 e 480 rxns The SMARTer Ultra Low Input RNA for Illumina Sequencing Components HV Cat No 634829 contains 5 x Cat No 634831 Not sold separately Box 1 5 x 96 ul 5x 5ul Box 2 5 x 96 ul 5 x 192 ul 5 x 384 ul 5 x 96 ul 5 x 96 ul 5 x 192 ul 5x 10 ml 5 x 528 ul 5x 10 ml 5x 10 ml SMARTer II A Oligonucleotide 24 uM Control Tota
Download Pdf Manuals
Related Search