User Manual - System Biosciences
Contents
1. with Next Gen Sequencing Z Complete exosome RNA Next Gen sequence i analytics solution for researchers interested in E identifying novel exosome associated RNA i biomarkers Abundance RNA type expression heatmaps and genomic mapping all included in service Exosome RNA Seq_and Mass _ Spec Bik sample prep kits Exosomes are the future of biomarkers in medicine The XRNA kits are specifically designed to make high quality exosome RNA Seq libraries for Illumina Next Gen sequencing using low RNA input amounts Discover exosome protein biomarkers using the XPEP Mass Spec peptide library sample prep kit G Shipping and Storage Conditions for Kits The XRNA kits are shipped on dry ice and should be stored at 20 C Avoid freeze thawing the reagents Shelf life of the product is 1 year after receipt if stored in 20 C 888 266 5066 Toll Free 650 968 2200 outside US Page 31 System Biosciences SBI User Manual ll References Munch EM Harris RA Mohammad M Benham AL Pejerrey SM Showalter L Hu M Shope CD Maningat PD Gunaratne PH Haymond M Aagaard K Transcriptome Profiling of microRNA by Next Gen Deep Sequencing Reveals Known and Novel miRNA Species in the Lipid Fraction of Human Breast Milk PLoS One 2013 8 2 e50564 Jiang K Hu Z Chen Y Jarvis JN Deep sequencing reveals differential small RNA expression in serum exosomes of children with juvenile idiopathic arthritis Arthritis Rheumatol 2014 Mar2. 66 Suppl 11 S230 Huang X Yuan T Tschannen M Sun Z Jacob H Du M Liang M Dittmar RL Liu Y Liang M Kohli M Thibodeau SN Boardman L Wang L Characterization of human plasma derived exosomal RNAs by deep sequencing BMC Genomics 2013 May 10 14 319 The UCSC genome browser and associated tools Kuhn RM Haussler D Kent WJ Brief Bioinform 2013 Mar 14 2 144 61 doi 10 1093 bib bbs038 FastQC A quality control tool for high throughput sequence data Simon Andrews http www bioinformatics babraham ac uk projects fastqc Fast gapped read alignment with Bowtie 2 Langmead B Salzberg SL Nat Methods 2012 Mar 4 9 4 357 9 doi 10 1038 nmeth 1923 ea utils Command line tools for processing biological sequencing data Erik Aronesty 2011 http code google com p ea utils Page 32 ver 3 063014 www systembio com XRNA Exosome RNA Seq Kits Cat XRNAxxxA 1 Cutadapt removes adapter sequences from high throughput sequencing reads Martin M http journal embnet org index php embnetjournal article view 200 479 Quality control and preprocessing of metagenomic datasets Schmieder R Edwards R Bioinformatics 2011 Mar 15 27 6 863 4 doi 10 1093 bioinformatics btr026 SeqPrep Tool for stripping adaptors and or merging paired reads with overlap into single reads http github com jstjohn SeqPrep Ultrafast and memory efficient alignment of short DNA sequences to the human genome Langmead B Trapnell C P
3. ensure enough read length to properly map a given RNA sequence to the genome While NGS is not as quantitative as qPCR expression patterns do clearly emerge of RNA sequence abundances when comparing across sample origins different conditions patients etc Sample exosome NGS data The final steps of the Maverix exosome RNA Seq analysis are abundance determination and differential expression analysis by DEseq Abundance levels for ncRNAs miRNAs tRNAs rRNAs lincRNAs piRNAs snoRNAs antisense transcripts coding genes and repeat elements LTR LINE SINE and tandem repeats are determined then a summary of reads overlapping each of these annotations in the reference genome is created using SAMtools and provided for visualization in pie charts using R a software environment for statistical computing and graphics 888 266 5066 Toll Free 650 968 2200 outside US Page 23 System Biosciences SBI User Manual Example RNA types and their relative abundance from Serum CSF Urine and Stem Cell Media exosomes to exons to introns fy Antisense to ncRNAs fg Antisense to repeat elements a snoRNAs a snoRNAs LINEs LTRs E Rfam ncRNA E Ee RefSeq exons RefSeq introns Urine Exosomes Stem Cell Media Exosomes The types and relative abundances of serum CSF and urine exosome RNAs appear to maintain and certain pattern between those biofluids The stem cell secreted exosome RNA types and abundances seem to be very diffe
4. highlighted in by the yellow box was excised for gel purification M is a 50 bp ladder and C refers to the custom ladder earl ial Adapter dimers do not want 41 Insert the gel cutter tool or excised gel fragments containing the gel slice into the gel breaker tube 42 Briefly spin the gel cutter and gel breaker assembly Make sure the gel slice is collected in the gel breaker tube Discard gel cutter 888 266 5066 Toll Free 650 968 2200 outside US Page 15 System Biosciences SBI User Manual 43 Add 50 ul of TE buffer with 0 1 Tween 20 to the gel breaker tube containing the gel slice If needed add additional TE buffer with 0 1 Tween 20 to cover the broken gel fragments 44 Centrifuge the gel breaker assembly in a bench top centrifuge at maximum speed approximately 13 000x g for two minutes at room temperature Ensure that all of the gel has moved through the holes into the collection tube 45 Elute the DNA by shaking the tube at 1000 rpm at room temperature for at least one hour The tube can be shaken overnight if desired 46 To collect the micro RNA library spin the gel mix at maximum speed approximately 13 000 xg for 2 minutes 47 With a P10 pipette gently remove eluate from gel mix Page 16 ver 3 063014 www systembio com XRNA Exosome RNA Seq Kits Cat XRNAxxxA 1 Library Validation 48 Use of an Agilent Technologies 2100 Bioanalyzer is recommended as a quality control analysi
5. ul of the supernatant Page 10 ver 3 063014 www systembio com XRNA Exosome RNA Seq Kits Cat XRNAxxxA 1 14 Repeat steps 10 and 11 once Remove and discard all residual supernatant after the second 80 ethanol wash 15 Air dry sample tube at room temperature for 15 minute or until the AMPure XP beads are dry 16 Remove sample tube from the magnetic stand Resuspend the dried AMPure XP beads in 8 5 ul of nuclease free water Incubate resuspension at room temperature for 2 minutes 17 Place the sample tube on the magnetic stand at room temperature for 5 minutes 18 Transfer 7 ul of the supernatant into a fresh 200 yl PCR tube 5 Adapter Ligation 19 Set up the following 5 Adapter Ligation reaction Reagent Volume ul 3 Adapter Ligated RNA from step 18 7 Mix D300 3 Mix E300 2 Total 12 20 Gently pipette mix thoroughly and incubate at 25 C for 1 hour and then place the tube on ice cDNA Synthesis 21 Pre heat the thermal cycler to 50 C 888 266 5066 Toll Free 650 968 2200 outside US Page 11 System Biosciences SBI User Manual 22 Set up the following cDNA Synthesis reaction on ice Reagent Volume ul 3 and 5 Adapter Ligated RNA from step 20 12 Mix F300 2 Mix G300 1 Total 15 23 Gently pipette mix thoroughly and incubate at 50 C for 1 hour and then place the tube on ice PCR Amplification 24 Set up the following PC
6. 00 6 Mix F300 3 Mix C300 7 Mix G300 4 Mix D300 8 Mix H300 The RNA Control is provided as a positive control to monitor the performance of the XRNA Sample Preparation kit The Control Library should contain a microRNA library peak at the 140 150 bp 888 266 5066 Toll Free 650 968 2200 outside US Page 3 System Biosciences SBI User Manual region The XRNA Sample Preparation Kit supports both single end and paired end sequencing on the Illumina platform The Illumina built in Read 1 primer is compatible with the XRNA libraries A custom primer must be used for Read 2 of paired end sequencing The Read 2 Primer is provided in 100 uM stock in 10mM Tris Set 2 Oligo Set Catalog Barcode Catalog Barcode XRNA101A 1 Sequence XRNA102A 1 Sequence PCR Primer PCR Primer Custom Ladder sae Custom Ladder E Barcode 1 ATCACG Barcode 9 GATCAG Barcode 2 CGATGT Barcode 10 TAGCTT Barcode 3 TTAGGC Barcode 11 GGCTAC Barcode 4 TGACCA Barcode 12 CTTGTA Barcode 5 ACAGTG Barcode 13 AGTCAA Barcode 6 GCCAAT Barcode 14 AGTTCC Barcode 7 CAGATC Barcode 15 ATGTCA Barcode 8 ACTTGA Barcode 16 CCGTCC Set 3 Gel Purification Kit 1 Gel Cutter Tool 2 Gel Breaker Tool Consumables Preparation The kit contains all necessary reagents to perform the experiment with the exception of common consumables and instruments Please make sure all equipment is available before starting this experiment s
7. 2 SBI System Biosciences XRNA Exosome RNA Seg Library Kit Cat XRNAxxxA 1 User Manual Store Kits at 20 C upon receipt A limited use label license covers this product By use of this product you accept the terms and conditions outlined in the Licensing and Warranty Statement contained in this user manual XRNA Exosome RNA Seq Kits Cat XRNAxxxA 1 Contents lee SITROGUCU ON eet tees atess fe a ehelaeiule aides te tabint eels 1 A EXOSOME OVELVICW eecceceeeeeeceeeeeee cee eeseaeeeeeeeseaeeesaeeneaeeteaes 1 B The XRNA kits for exosome RNA Seq NGS o ae 2 XRNA Procedures ccccceceeeeeeeeeeeeeaeeeeeeeseeeeeceaeeesaeeneneeeeaes 3 E Frequently asked QUESTIONS 0 ccc ceeeeeeeeeeeeeeeeeeteeeeeeeeeeeaeees 27 F Related Products and Services eneee 28 G Shipping and Storage Conditions for KitS ccceeeees 31 Il Referentes ienr aed ies clined Deeside ended eens 32 lil Technical Support cceccceceeeceseeeeeeeeeeeeeeeeaeeeeaeeseaeeeeaes 34 VII Licensing and Warranty information cccceeeeeees 34 l Introduction A Exosome Overview Exosomes are 60 180 nm membrane vesicles secreted by most cell types in vivo and in vitro These microvesicles are produced by the inward budding of multivesicular bodies MVBs and are released from the cell into the microenvironment following the fusion of MVBs with the plasma membrane Exosomes are extracellular nanoshu
8. R reaction in a fresh sterile 200 ul PCR tube on ice Reagent Volume ul cDNA from step 23 5 Mix H300 18 PCR Primer 1 Barcode Primer 1 Total 25 Only one of the barcode primers is used for each sample Page 12 ver 3 063014 www systembio com XRNA Exosome RNA Seq Kits Cat XRNAxxxA 1 25 Gently pipette mix thoroughly and amplify the samples in the thermal cycler using the following PCR cycling conditions 1 98 C for 30 seconds 2 18 20 cycles of i 98 C for 15 seconds ii 60 C for 15 seconds iii 72 C for 1 minute 3 72 C for 5 minutes 4 Hold at 4 C Library Purification 26 Determine the volume of TBE buffer needed and dilute 5X TBE Buffer to 1X for use in gel electrophoresis 27 Assemble the gel electrophoresis apparatus 28 Mix 2 ul of Custom ladder 140 160 500 bp with 2 ul of Hi Density TBE Sample Buffer 29 Optional Mix 2 ul of 50 or 100 bp DNA ladder with 2 ul of Hi Density TBE Sample Buffer 30 Add 2 5 ul of Hi Density TBE Sample Buffer to 25 ul of PCR product and pipet mix thoroughly 31 Load 25 ul of the PCR product into one well in the middle of the gel 32 To ensure precise excision of the target region load 2 ul of the Custome ladder and dye mix into two wells of the 8 PAGE gel 888 266 5066 Toll Free 650 968 2200 outside US Page 13 System Biosciences SBI User Manual bracketing each PCR product lane Refer to the gel Figur
9. ature for 30 minutes before use 2 Pre heat the thermal cycler to 70 C and pre heat another thermal cycler to 25 C if available 3 Denature the RNA Sample by assembling the following components in a sterile 200 ul PCR tube Reagent Volume pl exoRNA Sample 6 Mix A300 2 Total 8 888 266 5066 Toll Free 650 968 2200 outside US sl OM Clustering Sequencing exoRNAs Sequencing Barcode Clustering ri Primer Primer index Primer Page 9 System Biosciences SBI User Manual 4 Gently pipette mix thoroughly and incubate at 70 C for 1 minute and then place the tube on ice 5 Set up the following 3 Adapter Ligation reaction Reagent Volume ul Denatured RNA mix from step 4 8 Mix B300 2 Mix C300 6 5 Total 16 5 6 Gently pipette mix thoroughly and incubate at 25 C for 1 hour Ligation Product Clean Up 7 Vortex the AMPure XP beads until they are evenly resuspended 8 Prepare 80 ethanol for wash steps 9 Add 30 ul of AMPure XP beads to each sample Gently pipette mix thoroughly and incubate at room temperature for 15 minutes 10 Place the sample tube on the magnetic stand at room temperature for 5 minutes 11 Remove and discard 40 ul of the supernatant 12 Keep sample tube on the magnetic stand Gently add 100 ul of 80 ethanol into each sample tube without disrupting the beads Incubate at room temperature for 30 seconds 13 Remove and discard 95
10. d change and p values of serum exosome RNA expression Sample 1 vs Sample 2 Sample 1 vs Sample 3 Sample 2 vs Sample 3 v 2 s s i ee Si a Ee EE D et log2 fold chang i log2 fold change i log2 fold change cE Significant Significance Not Significant The example volcano plots above show how the exosome RNA differential expression can be visualized and used to quickly identify RNA biomarkers of interest Sample 1 is different from Sample 2 and 3 whereas Samples 2 and 3 are more similar 888 266 5066 Toll Free 650 968 2200 outside US Page 25 System Biosciences SBI User Manual Screenshot of interactive heatmap within the Maverix platform Mayers CRUK 31 DESeq Heatmap The heatmap above is displayed on the left hand side of its visualization with the integrated genome browser on the right In our example the first two columns are samples with the third column identifying the chromosomal location of features where chromosomes are classified by unique colors Hovering over a region of the heatmap brings up a tooltip as shown with information about the mapped exosomal component including name chromosomal position on the reference genome and differential expression values for each sample Clicking on the row for a feature of interest on the heatmap will bring up the associated region in the integrated UCSC Genome Browser for visualization of exosome components in their genomic context and wi
11. e in Step 40 for an example 33 Optional Load 2 ul of the 50 or 100 bp DNA ladder and dye mix into a separate well for additional position references 34 Run the gel for 65 minutes at 145V and immediately remove the gel from the apparatus Recover Purified Library 35 Prepare TE buffer with 0 1 Tween 20 Reagent Volume ul TE buffer 9 990 Tween 20 10 Total 10 000 36 Open the gel cassette and stain with 1ug ml ethidium bromide solution according to the manufacturer s instructions 37 Place the gel on a UV Transilluminator and observe the banding pattern see page 15 38 Alternative Stain gel with SYBR Gold according to the manufacturer s instructions and observe the banding pattern on a Dark Reader Transilluminator 39 Place the gel breaker tube into a sterile 1 5m microcentrifuge tube or 2 0 ml collection tube 40 Align the center of the gel cutter tool with the 160 bp band of the custom ladder on the sample lane to excise the libraries We recommend a broader gel excision range starting from 160 bp to Page 14 ver 3 063014 www systembio com XRNA Exosome RNA Seq Kits Cat XRNAxxxA 1 about 400 bp Press down firmly into the gel and excise the gel fragment This can be repeated to include larger band sizes up to about 400 bp Alternatively use a clean scalpel and excise the desired gel region Example of an 8 TBE gel stained with SYBR gold is shown below The library material
12. ecommend SBI s SeraMir kits see Table below Description Size Catalog SeraMir Serum Plasma Exosome RNA Purification only kit 5ml ExoQuick and 20 preps RA806A 1 20 exoRNA columns SeraMir Exosome RNA Purification only kit for Media Urine and CSF 10ml 10 preps RA806TC 1 ExoQuick TC and 10 exoRNA columns SeraMir Exosome RNA Purification Column kit 20 exoRNA columns eee cen Page 2 ver 3 063014 www systembio com XRNA Exosome RNA Seq Kits Cat XRNAxxxA 1 XRNA Kit Features e Wide dynamic range requires as little as 1 ng of exosome RNA input and up to 5 ug RNA input e User friendly workflow libraries can be prepared in a single day with less than one hour of hands on time e Comprehensive sample prep kit most components are supplied as ready to use mixtures which improves consistency and reproducibility e The XRNA kits come complete to create barcoded exosome RNA libraries compatible with Illumina HiSeq and MiSeq instruments e SBI sources the XRNA kit I components from SeqMatic SeqMatic LLC C XRNA Procedures Materials provided Each XRNA Sample Preparation Kit contains one set of core reagents 8 samples one set of 8 unique barcodes and a gel purification kit The core reagents and PCR barcode primers should be stored at 15 C to 25 C The kit is designed to be stable for up to one year after the shipping date Set 1 Core Reagents 1 Mix A300 5 Mix E300 2 Mix B3
13. ee next table Page 4 ver 3 063014 www systembio com XRNA Exosome RNA Seq Kits Cat XRNAxxxA 1 1 5 ml nuclease free microcentrifuge General lab supplier tubes Thermal cycler General lab supplier Beckman Coulter Genomics A63881 888 266 5066 Toll Free 650 968 2200 outside US Page 5 Agencourt AMPure XP beads System Biosciences SBI User Manual SYBR Gold Nucleic Acid Gel Stain Optional LIFE Technologies S 11494 Tube shaker or thermal mixer General lab supplier 2100 Bioanalyzer Agilent Technologies Agilent Technologies 5067 4626 High Sensitivity RNA and DNA chips Best Practices e Always wear gloves and use sterile technique e Setup reactions using sterile non stick nuclease free tubes e Place samples and reagents on ice at all times and avoid extended pauses e Reagents should be prepared using RNase free components e Prepare an extra 10 mixture when running multiple samples e Avoid repeated freeze thaw cycles Page 6 ver 3 063014 www systembio com XRNA Exosome RNA Seq Kits Cat XRNAxxxA 1 RNA Input This protocol has been optimized using approximately 1 to 10 ng of purified exosome RNA Typical exosome RNA ranges in very small sizes 20 30 nt like miRNAs and have a peak abundance of exoRNAs in the 150 250 nt size range Due to this small exoRNA size range and low abundance typical UV based RNA quantitation methods like NanoDrop may not be sensitive enough t
14. exo ngs Isolating Exosome using ExoQuick ExoQuick TC and Exo Flow Immunocapture kits Description Size Catalog ExoQuick Serum exosomes 75 rxns EXOQ5A 1 ExoQuick Plasma Exosome prep 75 rxns EXOQ5TM 1 Thrombin Plasma Exosome prep 100 rxns TMEXO 1 ExoQuick Serum exosomes 300 rxns EXOQ20A 1 ExoQuick TC for Tissue Culture Media 10 rxns EXOTC10A 1 ExoQuick TC for Tissue Culture Media 50 rxn EXOTC50A 1 Exosome isolation protocols using ExoQuick reagents Combine your biofluid sample containing exosomes with ExoQuick or ExoQuick TC using the guidelines shown in the Table below Mix the ExoQuick precipitation reagent with the biofluid sample by inversion and place at 4 C for 30 minutes to overnight then 888 266 5066 Toll Free 650 968 2200 outside US Page 19 System Biosciences SBI User Manual recover the exosomes in a pellet with a low speed spin Please refer to the ExoQuick or ExoQuick TC User manuals for more details Recommended amounts of exosomes provided in Table Resuspend Volume to exosome use in pellet Exo Flow 2 ml 500 uL PBS 100 pL rxn Spinal fluid 2 ml 500 uL PBS 100 pL rxn Culture media 500 uL PBS 100 pL rxn Sample i ExoQuick Bioflui giad volume TC volume Resuspend Volume to ExoQuick exosome use in Exo pellet Flow Serum 500 pL 120pL alles 100 pL rxn Ascites fluid 500 pL 120 uL eB 100 pLirxn Amount of exosomes to use The number of exosomes in a g
15. ia from cells in culture you should grow your cells in the absence of bovine FBS SBI offers bovine exosome depleted FBS for this purpose cat EXO FBS 50A 1 Urine and CSF samples should be pre spun at 3 000 xg to pellet cellular debris prior to exosome isolation with SBI s ExoQuick TC cat EXOTC10A 1 NOTE ExoQuick and ExoQuick TC for exosome isolation purposes are not provided in the XRNA kits and can be purchased separately The following ExoQuick products are recommended for exosome concentration prior to Exo Flow purification XRNA Library Workflow Overview The XRNA kits have a user friendly workflow that allows the preparation of exosome RNA Illumina NGS libraries for sequencing in a single day with minimal hands on time All reagents are supplied as optimized ready to use mixtures The XRNA kits include specialized plastics and gel cutters to streamline gel purification of amplified NGS libraries The kits are fully compatible with Illumina sequencing platforms including HiSeq MiSeq and Genome Analyzer Il Page 8 ver 3 063014 www systembio com XRNA Exosome RNA Seq Kits Cat XRNAxxxA 1 SeqMatic Se P OH 3 Adapter Ligation l pe P T 3 FT 5 Adapter Ligation l S A 3 eS cDNA Synthesis 1 3 EZ amp 8 8s a 3 PCR Amplification ayaa i i 5 Library Purification eee mer XRNA Sample Library Prep Protocol 3 Adapter Ligation 1 Allow Mix C300 to equilibrate to room temper
16. iven biofluid will vary depending upon the sample itself There are abundant levels of exosome in serum less in cell culture medium and urine Use the guidelines in the tables above as a starting point Sample Biofluid volume Page 20 ver 3 063014 www systembio com XRNA Exosome RNA Seq Kits Cat XRNAxxxA 1 D Sample XRNA exosome RNA Seq data PE 2x 75 bp DATA PREPROCESSING examples Sequence QC Pre trimming Bese EE z 2 123456789 11 13 15 17 19 21 23 25 27 29 31 33 3S 37 39 41 43 45 47 49 51 S SS S7 59 G1 63 65 67 69 71 73 75 Position in read bp Sequence QC Post trimming 123456785 11 13 15 17 19 21 23 2 27 29 31 33 35 37 39 41 43 45 47 49 51 53 55 57 59 6l 63 65 67 69 71 73 75 Position in read bp Example Mapping Statistics summary The improved set of sequence reads are merged if needed using 888 266 5066 Toll Free 650 968 2200 outside US Page 21 System Biosciences SBI User Manual SeqPrep then mapped to the reference genome using Bowtie an ultrafast memory efficient short read aligner followed by generation of a mapping rate summary chart for review Using the open source software BEDTools and SAMtools read alignment and read coverage tracks are generated and deployed to the genome browser 0 v 4 vt HEJ c m 5m x Filtered Out S g rerainea Unmapped N E A 1 Retained Mapped Ss Zg v S1 S2 s3 S4 S5 S6 Sequencing read mapping rate The char
17. o properly measure the purified exoRNA concentration We recommend using an Agilent Bioanalyzer 2100 system with the Agilent RNA 6000 Pico Kit which is optimized for the analysis of low concentrated RNA samples down to 50 pg uL of total RNA or 250 pg uL of microRNA An example of purified serum exosome RNA 1 ul of 15 ul total exoRNA from 500 ul starting serum is shown below SBI s ExoQuick and SeraMir kits were used to purify the exoRNA GEL ELECTROPHEROGRAM g we Fu 4 8 Y 7 Longer exoRNAs 6 l e 5il z T Longer F H exoRNAs 150 miRNAs Zit w00 H s0 6 ae 60 aap 40 M miRNAs 20 al l Small RNA Concentration pg ul 589 4 M 4 4 20 40 60 80 100 150 200 250 nt Serum exosomes tip Use ExoQuick precipitation twice on a serum sample to remove some co purifying serum proteins To do this take 500 ul serum add 120 ul ExoQuick and incubate at 5 C for 30 minutes Spin the tube for 3 minutes at highest speed 888 266 5066 Toll Free 650 968 2200 outside US Page 7 System Biosciences SBI User Manual Discard supernatant Resuspend the exosome pellet in 250 ul 1x PBS and add 60 ul ExoQuick Incubate at 5 C for 30 minutes and spin for 3 minutes at maximum speed to pellet the exosomes These are now ready for exoRNA purification If you have plasma samples please defibrinate using SBI s cat TMEXO 1 kit Media Urine CSF exosomes tip For studying exosomes in med
18. op M Salzberg SL Genome Biol 2009 10 3 R25 doi 10 1186 gb 2009 10 3 r25 BEDTools a flexible suite of utilities for comparing genomic features Quinlan AR Hall IM Bioinformatics 2010 Mar 15 26 6 841 2 doi 10 1093 bioinformatics btq033 The Sequence Alignment Map format and SAMtools Li H Handsaker B Wysoker A Fennell T Ruan J Homer N Marth G Abecasis G Durbin R 1000 Genome Project Data Processing Subgroup Bioinformatics 2009 Aug 15 25 16 2078 9 doi 10 1093 bioinformatics btp352 Differential expression analysis for sequence count data Anders S Huber W Genome Biol 2010 11 10 R106 doi 10 1 186 gb 2010 11 10 r106 R A language and environment for statistical computing R Development Core Team 2008 R Foundation for Statistical Computing Vienna Austria ISBN 3 900051 07 0 http www R project org 888 266 5066 Toll Free 650 968 2200 outside US Page 33 System Biosciences SBI User Manual lll Technical Support For more information about SBI products and to download manuals in PDF format please visit our web site hitp www systembio com For additional information or technical assistance please call or email us at System Biosciences SBI 265 North Whisman Rd SBI Mountain View CA 94043 Phone 650 968 2200 888 266 5066 Toll Free Fax 650 968 2277 E mails General Information info systembio com Technical Support tech systembio com Ordering Information orders
19. pinal fluid Purified human cancer exosomes and and mouse dexosomes Use for RNA Protein analysis calibration standards and engineering cargo for delivery to target cells All exosomes are characterized by NanoSight for size intactness and concentration as well as tested to be CD63 positive by Western blot analysis The purified exosomes are provided frozen with gt 1x10 6 exosomes 50 ug protein Exosome Cargo Labeling Fluorescently label exosome cargo Label endogenous exosome RNAs Red and internal exosome Proteins Green to monitor exosome cargo delivery to cells in real time Page 28 ver 3 063014 www systembio com XRNA Exosome RNA Seq Kits Cat XRNAxxxA 1 RabSb Beads Exo FITC PLUS Exosomes Immunopurify Exosomes and use with FACS Selectively capture distinct subpopulations of intact exosomes based on a particular surface i marker and sort by FACS Flow Exometry 9 Choose from the following tetraspanin annexin adhesion fusion and immune presentation targets or customize your own capture system Singlets CD9 e CD31 CD63 CD81 3 Rab5b HLA G ey A Exosome RNA Seq_ and Mass _ Spec sample prep kits Exosomes are the future of biomarkers in medicine The XRNA kits are specifically designed to make high quality exosome RNA Seq libraries for Illumina Next Gen sequencing using low RNA input amounts Discover exosome protein biomarkers using the XPEP Mass Spec peptide librar
20. rent from patient biofluid exosomes IncRNA Tandem repeats Unannotated lincRNA microRNA piRNA rRNA scaRNA tRNA tRNA like Differential expression analysis across samples The differences in expression of ncRNA antisense transcripts and repeat elements between samples can be calculated based on how many times a given RNA sequence is read and calibrated to the total number of reads mapped for that given sample Visual representation of the analysis results are provided in the online Maverix platform including volcano plots showing an overview of significantly differentially expressed genes as well as interactive tabular and heatmap views linked to the integrated UCSC Page 24 ver 3 063014 www systembio com XRNA Exosome RNA Seq Kits Cat XRNAxxxA 1 Genome Browser The Maverix Exosome RNA seq Analysis produces outputs that include read alignment and coverage tracks for the genome browser annotations to the reference genome as well as abundance determination and differential expression analysis The Maverix Analytic Platform allows researchers to visualize their data and analytic results using an integrated UCSC Genome Browser automatically configured for their specific organism of interest With access to data in the UCSC Genome Browser directly from the platform researchers can add custom tracks to the browser securely surf their data and easily share or publish their results Sample Volcano Plots showing fol
21. s of your sample library Use 1 ul of resuspended construct from step 45 on a High Sensitivity DNA chip to check the size purity and concentration of the sample 7000 2000 Sample Agilent ee Bioanalyzer 2100 500 High Sensitivity DNA 400 Assay 300 Electrophoresis File 200 p Run Summary shown 100 E eel to the right 35 ee es ete L 123 4 5 6 FU 150 a n 100 J i F L ese ii _ a Sd eel O S E E B O B S 35 150 300 500 1000 10380 bp Sample Pooling The XRNA exosome RNA NGS Sample Preparation kit is capable of multiplexing up to 16 samples into a single lane of an Illumina flow cell Multiplexing of 6 to 8 libraries is recommended for each MiSeq v3 sequencing run While processing multiple samples in parallel use a unique barcode primer for each sample at the PCR step Samples can be pooled before or after the library purification step 888 266 5066 Toll Free 650 968 2200 outside US Page 17 System Biosciences SBI User Manual NGS Data Analysis Overview The Exosome RNA seq Analysis See Section D Sample XRNA exosome RNA Seq data initiates with a data quality check of the input sequence using FastQC an open source quality control QC tool for high throughput sequence data FastQC runs analyses of the uploaded raw sequence reads that reveal the quality of the data and inform the subsequent preprocessing steps in the analysis Following the initial assessment Bow
22. systembio com Vil Licensing and Warranty information Limited Use License Use of the XRNA Kits i e the Product is subject to the following terms and conditions If the terms and conditions are not acceptable return all components of the Product to System Biosciences SBI within 7 calendar days Purchase and use of any part of the Product constitutes acceptance of the above terms The purchaser of the Product is granted a limited license to use the Product under the following terms and conditions Page 34 ver 3 063014 www systembio com XRNA Exosome RNA Seq Kits Cat XRNAxxxA 1 e The Product shall be used by the purchaser for internal research purposes only The Product is expressly not designed intended or warranted for use in humans or for therapeutic or diagnostic use e The Product may not be resold modified for resale or used to manufacture commercial products without prior written consent of SBI e This Product should be used in accordance with the NIH guidelines developed for recombinant DNA and genetic research Purchase of the product does not grant any rights or license for use other than those explicitly listed in this Licensing and Warranty Statement Use of the Product for any use other than described expressly herein may be covered by patents or subject to rights other than those mentioned SBI disclaims any and all responsibility for injury or damage which may be caused by the failure of the b
23. th public datasets The identities of the microRNAs in the sample figures above have been purposefully hidden with a dark blue box For more information about the Maverix analytics platform visit www maverixbio com Page 26 ver 3 063014 www systembio com XRNA Exosome RNA Seq Kits Cat XRNAxxxA 1 E Frequently asked questions How many reads do need per sample How many libraries can multiplex on a MiSeq Should do single or paired end reads How long does a MiSeq run take What file format does it generate How much PhixX Control do need for the MiSeq sequencing run 888 266 5066 Toll Free 650 968 2200 outside US Cellular RNA is complex requires 300M reads per sample Exosomes have 1 of that complexity need 3 5M reads per sample MiSeq v3 produces 25M reads If you want 3 5M reads sample pool 6 libraries We recommend paired end 2x 75bp NGS runs Gives greater confidence of RNA sequence identity The typical MiSeq run is 18 hours You will only need the FASTQ files A minimum of 5 PhiX Control spike in is recommended Page 27 System Biosciences SBI User Manual F Related Products and Services SBI offers a number of exosome research products Review them here http Awww systembio com exosomes Isolate Exosomes with ExoQuick and ExoQuick TC One step Exosome Isolation for Serum and Plasma Tumor Ascites Fluid Follicular fluid Ye Tissue Culture Media Urine S
24. tie2 is used to map the spike in DNA before the analysis moves to the trimming and filtering steps where RNA seq reads are preprocessed to improve the quality of data input for read mapping The open source tools used for trimming of adapters are FastqMcf part of the ea utils package and cutadapt with PRINSEQ used in the quality filtering step Maverix exosome RNA Seq 1 information link verix http Awww maverixbio com platform Bi s Exo NGS Custom Services SBI and Maverix Biomics have teamed up to provide a complete analytics solution on deep sequencing data of exosome associated RNAs The analysis service includes library sequence quality control metrics data analysis for relative RNA abundance and identity differential expression analysis and visualization of the data in a cloud based private UCSC Genome Browser Simplify and accelerate your exosome RNA biomarker discovery with the advanced bioinformatics analysis included in SBI s Exo NGS service Visit www systembio com exo ngs Page 18 ver 3 063014 www systembio com XRNA Exosome RNA Seq Kits Cat XRNAxxxA 1 DISCOVER illumina Exosome RNA Illumina NGS sequencing and analysis service Isolates exosomes Mail SBI samples Purifies exoRNA serum urine media Makes bar coded Illumina Receive raw data and analysis libraries report Runs MiSeq HiSeq Identify novel exoRNAs Performs data analysis Get your next major grant wwwasystembio com
25. ts show the percentage and number of reads respectively for trimmed mapped and unmapped reads for each of the samples In most NGS runs adapter dimers form during amplification and barcoding These adapter sequences are filtered out and removed from the exoRNA sequences of interest before mapping to the genome of choice i e Human Mouse Rat etc A typical NGS sequence data set may filter out 50 of the reads due to QC and or adapter sequences present Page 22 ver 3 063014 www systembio com XRNA Exosome RNA Seq Kits Cat XRNAxxxA 1 What should I expect Cellular RNA Seq versus exoRNA Seq A typical cellular RNA content is extremely complex with miRNAs piRNAs tRNAs mRNAs IncRNAs rRNAs and other ncRNAs These types of cellular RNA Seq NGS experiments require about 300M reads per sample to obtain a depth worth analyzing In contrast exosome are tiny nanovesicles averaging about 100nm in diameter The cargo of an exosome is comprised of proteins and RNAs Due to these size constraints the exosome RNA complexity and diversity is estimated to be that of about 1 of a cellular RNA diversity Exosome RNA population lengths are small to begin with 250 nt and smaller as seen in the Agilent Bioanalyzer RNA chip analysis We suggest targeting approximately 2 5 million reads per sample for these reasons to achieve a depth where expression differences can be easily observed We also recommend performing paired end 2x 75 bp reads to
26. ttle organelles that facilitate communication between cells and organs Exosomes are found in blood urine amniotic fluid breast milk malignant ascites fluids and contain distinct subsets of RNAs and proteins depending upon the cell 888 266 5066 Toll Free 650 968 2200 outside US Page 1 System Biosciences SBI User Manual type from which they are secreted making them useful for biomarker discovery SBI has engineered tools and provides services for exosome proteomic Mass spec analysis and next generation sequencing of exosome RNA to accelerate the study of exosomes exosome protein and exosome biomarkers B The XRNA kits for exosome RNA Seq NGS The XRNA RNA Seq Sample Preparation Kits are sourced from SeqMatic LLC The system offers a high sensitivity solution for generating exosome RNA including miRNA libraries from low concentration RNA samples These kits enable the discovery and profiling of miRNAs from various organisms and tissues via the Illumina sequencing platform The unique XRNA reagents and workflow have been developed for simplicity and reproducibility without sacrificing quality or yield The kits work with exosomes isolated using ultracentrifugation as well as using ExoQuick serum plasma ascites samples or ExoQuick TC cell media urine spinal fluid or immunopurify specific exosome subpopulations using SBI s Exo Flow IP kits The isolated exosomes are then lysed and the RNA purified using spin columns we r
27. uyer or any other person to use the Product in accordance with the terms and conditions outlined herein Limited Warranty SBI warrants that the Product meets the specifications described in this manual If it is proven to the satisfaction of SBI that the Product fails to meet these specifications SBI will replace the Product or provide the purchaser with a credit This limited warranty shall not extend to anyone other than the original purchaser of the Product Notice of nonconforming products must be made to SBI within 30 days of receipt of the Product SBI s liability is expressly limited to replacement of Product or a credit limited to the actual purchase price SBI s liability does not extend to any damages arising from use or improper use of the Product or losses associated with the use of additional materials 888 266 5066 Toll Free 650 968 2200 outside US Page 35 System Biosciences SBI User Manual or reagents This limited warranty is the sole and exclusive warranty SBI does not provide any other warranties of any kind expressed or implied including the merchantability or fitness of the Product for a particular purpose SBI is committed to providing our customers with high quality products If you should have any questions or concerns about any SBI products please contact us at 888 266 5066 2014 System Biosciences SBI All Rights Reserved Page 36 ver 3 063014 www systembio com
28. y sample prep kit 888 266 5066 Toll Free 650 968 2200 outside US Page 29 System Biosciences SBI User Manual FBS Exosomes Culture Cells in Exosome depleted FBS Removed Study exosomes from cultured cells and not from CA E bovine exosomes in FBS itself Exo FBS has been stripped of bovine exosomes yet supports Exa Fas i Exo t BS robust growth of cells in culture Soa io D Verify Exosome Recovery with Antibodies KDa mw S OS 3 and Antibody Arrays 100 Track exosomes by Western blots and Antibody 70 Arrays using well characterized exosome protein 55 markers Verify exosome recoveries after isolation with ExoQuick or ultracentrifugation 35 using validated antibodies and arrays 25 Quantitate Exosomes with ELISAs ExoELISA _CD9 Serum Plate 3 pom Exo ELISAs measure the levels of exosome e particles with antibodies to detect CD9 CD63 or CD81 Highly sensitive and quantitative assays oy a in a convenient 96 well format with validated sensitive and Accurate Quantitation exosome standards j Amplify ExoRNA for Arrays M Un Amp uig 33 EH a i Page 30 ver 3 063014 www systembio com Amplify Exosome MicroRNAs with SeraMir for qPCR and microarrays Purify exoRNAs with SeraMir columns and covert to cDNA for microRNA qPCR arrays or amplify exoRNAs for microarrays analysis XRNA Exosome RNA Seq Kits Cat XRNAxxxA 1 Discover Novel Exosome RNA Biomarkers Put your Data into Context
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