Attagraph User Manual
Contents
1. RAA AAA AAA AA AAA AANER AAA RAA A REN A AAA A AAA AA SIGNAL TRANSDUCTION TRANSCRIPTION FACTORS DRUG DISCOVERY ATTAGRAPH Reader Users Manual Cap E File Calibration oe ee A Sox Ten mg Tcfif cat 5 sat E Box aita gene You are using Factorial 30A ATTAGRAPH Reacher wv 1 4 6 amp Ze 908 0 9 8 9 0 WW 90 9 0 WW WW 9 9 WW 9 9 Wi IW 0 9 9 sg BA RA AA BR ARA AAA RK AAA AAA AR RAA AAR RA RAA ARA AAA ARA RAA A Table of Contents Table 1 Glossary for ATTAGRAPH toolbar buttons Tae ee ee eee 3 Analysis of Data overview and computer requirements 4 L ATTAGRAPH installation process 0 000 002 e eee 5 ll Changing version of Factorial in use 1 2 0 0 0 0 eee 6 Ill Calibration of the ATTAGRAPH Software u un auaauua 8 IV Analyzing experimental data 15 Table 2 Standard Peaks Table 18 Trouble SNOOUNG BEER 22 Appendix A Settings used on the ABI 3130 XL Genetic analyzer 26 Appendix B End user license agreement n a nn annaa anaa ER Table 1 Toolbar Glossary shows reporter and standard peaks data in tabular format 73 Show radar graph of TF s activities d Expand the vertical scale of the profile Expand the horizontal scale of the profile LI Reset the horizontal and vertical scales of the profile M Select or deselect to show or hide the Blue reporter peaks D Select or deselect to show or hide the red size standard peaks Chang2. 1 077 19901 8036 1 292 3064 81355 0 615 34595 8241 169 23112 6344 1 400 15387 8500 0 969 19932 68584 1 230 22343 B666 1 382 15991 8798 1 000 27201 9091 1 646 13446 9329 0 807 25595 9681 1 455 20624 9917 1 163 30387 10271 1 676 12929 10542 0 729 15419 11725 0 794 10679 11656 0 553 i7461 12105 0 881 10145 12229 0 518 10454 12476 0 529 1f647 12600 0 851 E 1 LU 1 1 1 1 1 1 1 1 1 1 LU 2 1 LU 1 1 Copy all data SKI 1 076 1 662 1 803 1 881 64 035 20 945 ZA foo Lvl 5 068 3 513 1 326 14 625 1 911 1 182 1 567 0 963 170 980 20 Z 0 559 1 145 3 159 1 000 0572 0 307 5 574 Baal 3 170 0 950 2 348 1 575 0 937 0 760 0 319 16 761 Relative Peak Height Specific TF Activity TF Name TGFERE TATA 1 TCF b cat PPRE GRE OP 1 ISRE NF kB Fox Mbp CRE Dt SRE ERE ck HSE SREBP pos BRE Fax HIE a TATA 2 TATA 3 TAT A 4 ETS NRF 1 GAT A C EBP Mib Db 2 RARE TATA 5 Fox S5Sox9 Spi Copy TF activities 9 Once you have checked to make sure all calibration peaks have positive values and there are no un named peaks you may close these two windows and proceed to analyzing your experimental data 14 IV Analyzing experimental data 1 Go back to main frame window and select file open to browse for an experimental data file Select the file you want and click open as shown below 2008 01 09 atesi FOS fea Experimental My Recent 2008 01 09_plat
3. and load the FSA file into ATTAGRAPH Cause Too much PCR product was used for the labeling reaction and parasite peaks have occurred 24 Solution Recheck the amount of PCR product against the mass ladder If there is too much PCR product present then use 1 20 of the reaction for the labeling reaction Problem 7 Size standard signal is atypical See representative example below SE TRAST CEPTS FORTS TROT PPR WEE a AE PSEA TD PEP Compared to the normal profile below Cause Most of the named reporter peaks are missing Solution adjust threshold Cause Sample isn t clean Solution purification of PCR product is necessary with the proteinase K step Solution Replace defective reagents or array and re run your samples For Technical Support please contact us at info attagene com or call 888 721 2121 St Appendix A Settings used on the ABI 3130 XL Genetic analyzer Electrophoresis machine for fragment analysis of the FACTORIAL Reagent Mame Value Range o 18 65D0gC Hoh FEIL Vol e 65004 6500 38000 steps y Current stability el 50 ei 02000 Amps PreRun_Voltaqe el 150 si 0 15 Kolts Fre Fun Time el 15 1 1000 sec S Injection_Voltage el l2 e 1 15 Kols 8 Injectian_Time el 23 1 5600 sec E Voltage Number Ot steps 20 e 1 100 nk z Voltage step Interval el 15 ei 1 60 sec 8 Data_Delay_Time el DU ei 1 53600 sec 2 Run_Yoltage ei JUDD 0 15 kVolts un Time el 2000 sUU
4. from a capillary electrophoresis run To do so select file open then choose a file and click open JAttaGraph ota File Calibration Open Open F54 File and show graphics TGF Preferences AC P deems Tefip cat E Box PPR Exit i EA j Open Lookin Calibration files EI P Cal 2008 01 09_plate512_F09 fsa H Calibration file My Recent ie 2008 01 09_plate512_G09 fsa Documents My Documents A My Computer B E File name 2008 01 09_plate512_E09 fsa My Network Places Files of type FSA or ATG Files ise 2 Once the calibration file is open you should see a profile like the example below The profile below shows peaks resolved according to size along the horizontal or x axis and the heights determines the relative abundance in fluorescent units y JAttaGraph Z Calibration data 2008 01 09_plate512_E09 fsa ogg Es gt ol O m m Blue peaks are calibration peaks Red peaks are size standard peaks Vertical axis Represents fluorescent units Horizontal axis represents size It is suggested that the calibration is completed for every capillary electrophoresis run to ensure the correct name is assigned to each reporter peak 3 You will need to zoom in and check the threshold line and make sure it overlaps all calibration peaks to distinguish from background or noise peaks You can adjust this line by hovering over the grey li
5. the fsa file to save on confusion b A csv file which saves the raw capillary electrophoresis values and could be used with other spreadsheet applications that require this type of file This file shows the data in the raw form and has 4 columns for each of four channels blue green black and red The blue TF activity is column A while the red size standard is column D Columns B and C are for other channels that are not currently used Each scan from the capillary electrophoresis machine is assigned a fluorescent value You can open this data in Excel and draw graphs change scales Below is an example of a CSV file opened in Excel and line graphs drawn for the corresponding data D LG D E F G H i J K L Mi N oO Example of TF activity In Excel O TF reporter peaks mm Size standard peaks Fluorescence units 21 Troubleshooting Problem 1 Small or No peak heights for the Reporter blue peaks between the vertical gray lines but your standard red peaks look fine Cause The sample was not added into the master mix plate before loading onto the capillary electrophoresis machine Solution Rerun the samples with both the size standards and the labeled experimental sample present Cause Not enough PCR product in your primer extension reaction Solution Repeat the PCR step VI and increase the number of cycles up to 35 cycles Make sure that you have used a Mass Ladder to correctly assess the amount o
6. 1 20 359 59 808 9281 8548 Pax6 EA 21 364 53 932 10915 8633 HIF 14 E 22 369 01 324 3608 8710 TATA 24 23 376 74 928 10981 8843 TATA E 24 394 07 om 29544 M4 TATA E 26 407 93 u 4982 9380 ETS 27 26 428 36 oo on 9733 NRF EJ o 442 19 617 7839 9972 GAT 29 28 464 m 17772 10331 GER EJ 29 480 73 445 5852 10604 Myb EI 30 553 66 461 6367 11807 AP 2 32 31 562 08 661 9682 11940 RARI 33 321 677 97 627 9944 12192 TATA EI 33 585 98 464 6920 12319 Foxa E 34 602 15 506 7568 12674 Soxg E 36 610 31 1119 18117 12699 Spt ER ES M 4 gt 1 Sheet1 Sheet2 Sheet3 19 8 You can also view your data graphically in our software to view the data in Radar graphs within our software select the Attagraph viewer button as shown below Bab ole m REATA 1 TCFib cat The button toggles between IF activity and relative height of corresponding reporter peaks The button is for scale and can be toggled from linear to log base4o 20 9 Not only can you look at your graphs but you can save the raw data in two formats a An atg file this is the recommended way to save the raw data and it will have experimental data attributes attached so one may look at the data later and have all relevant data from the experimental run Once you have data saved as an atg file you can open data without worrying about Factorial version used or calibration files We recommend naming the atg file the same as
7. 14000 sec 26 Appendix B End user license agreement This End User License Agreement EULA is a legal agreement between you either an individual or a single entity and Attagene Inc This software is the copyright of Attagene Inc and is made available for analyzing data generated during the Factorial assay This software is not intended for use with any other application This software can t be redistributed whatsoever and is not for commercial use This software is solely for use by purchaser of factorial reagent kits and not for use with any other application This software can not be redistributed in the original or modified form Not responsible for incompatibility issues with the JAVA interface Java is a trademark of Sun Microsystems YOU AGREE TO BE BOUND BY THE TERMS OF THIS EULA BY LINKING TO OR OTHERWISE USING THE SOFTWARE IF YOU DO NOT AGREE TO THE TERMS OF THIS EULA YOU MAY NOT USE THE SOFTWARE AND YOU SHOULD PROMPTLY CONTACT ATTAGENE Inc FOR INSTRUCTIONS ZI
8. 5 1 814 0 756 1 940 1 479 1 235 1 379 1 424 1 050 1 060 1 089 LIZ 1 257 0 613 2 168 1 370 0 970 1 209 1 376 1 000 1 588 0 772 1 398 24128 10044 1 138 34791 10405 1 599 14992 10681 0 691 18272 11890 0 783 12097 12024 0 523 19550 12278 0 819 11194 12405 0 476 11572 12661 0 484 4787 4275 4581 4659 4648 5057 5027 5867 5628 6713 6390 7572 7380 8437 8184 9303 9002 10171 0 475 9559 10576 0 491 10239 11000 0 507 12626 11825 0 568 LL zo 0 377 0 359 0 360 0 366 0 387 0 415 0 450 0 464 B TATA 1 TCF b cat PPRE GRE AP 1 ISRE NF kB Fo xbpi CRE AhrE ARE ERE Oct HSE p53 BRE Standardi Standard2 Standard3 Standard4 StandardS Standard6 Standard Standards Standard9 Standard10 Standard11 Standard12 i Copy TF activities Close Table 2 Standard size table TF activit Blue peaks Standard 13 om Size standard Red peaks pi v Note There should be 35 blue TF activity peaks and 14 red standard size peaks that you should see in this view Note The standard size peaks values should match the values in Table 2 above Note If you do not see the red size standard peaks you may have deselected them in the graph this would be the case for blue TF activity peaks if you had deselected them as well 18 You can now select all data or only specific TF activities for copy and paste into Excel To copy all data click on copy all da
9. To change the FACTORIAL version 1 Select file from main menu and choose preferences E Open preferences dialog e You are using Factorial 30A ATTAGRAPH Reader v 1 4 8 2008 2 From the preferences pop up menu choose the drop down menu that corresponds to the FACTORIAL TV you are using then click ok A N ow you should see which FACTORIAL version that is in use as circl sJUAttacraph File Calibration ed below Jes ATTAGRAPH Reader v 1 4 8 2008 lll Calibration of the ATTAGRAPH Software After initializing and registering the ATTAGRAPH Reader software FACTORIAL data can be analyzed Before the experimental data can be correctly processed the ATTAGRAPH Reader software should be calibrated This calibration will allow the software to correctly interpret the FACTORIAL data generated by the capillary electrophoresis machine Note Currently our software only reads runs generated from ABI capillary electrophoresis machines Note calibration must be performed to compensate for the differences intrinsic to a specific capillary electrophoresis machines with regards to sizing of the DNA fragments 1 To calibrate the ATTAGRAPH Reader software you need to load a file from one of the calibration standards that were run in step 10 of the FACTORIAL H protocol This is an fsa file
10. e between use of logarithm scale or linear scale Change between TF s activity or relative peak values Attagraph Reader software is required for analysis of the Factorial data The software is provided as a part of the Factorial30 transcription reporter system It may be linked from our webpage at Attagene com Note Please read the end user license agreement in appendix B of manual One will see this agreement during initialization of software Analysis of Data ATTAGRAPH Reader is a computer program that analyzes capillary electrophoresis fragment analysis files generated by the FACTORIAL assay The essence of the FACTORIAL technology is a library of uniformly constructed Reporter Transcription Units RTUs Analysis of FACTORIAL data with ATTAGRAPH Reader will allow the user to determine the activity of each RTU and compare it to the RTU activity after a desired treatment In order to analyze this data and interpret the results of the FACTORIAL assay you must e Calibrate the software to a specific capillary electrophoresis machine e Open FACTORIAL data files in the form of raw fsa files for analysis e Analyze the FACTORIAL data e Export the results to an Excel spreadsheet for further analysis e Save data as an ATG file or a CSV file for storage and retrieval ATTAGRAPH TN will only work properly if supplied with raw fsa files from an ABI capillary sequencer Some sequencing facilities may ha
11. e512_G09 Fsa Documents 3 Desktop My Documents E My Computer 8 File name ID Oper My Network f EN Places Files of type FSA or ATG Files K cance 15 2 When data opens you will see a graph similar to the one below This is your experimental data 4 JAttaGraph C Documents and Settings Admin 01 THERALOGICS001 000Weskto OJEJ Reporter peaks Standard size peaks threshold example II od IHI Size standard IIIT peaks example Calibration peaks Threshold line Lill dii l ni il id id UL y Motta dled HEISER E E EA SCHER EISES UNE OF IPES E ES iat EMS Uer Background or noise peaks One can select or deselect the red size standard peaks or blue reporter peaks to view them separately 3 Unlike a calibration sample in the experimental sample some TF reporter peaks may not be present due to low corresponding TF activity We recommend placing the threshold as low as possible but not below 50 fluorescent units Check the value of the lowest TF activity peak crossed by the threshold and make sure that it s not below 50 fluorescent units 16 4 One should also look at the under digested fraction peak which is the 1 blue peak after the horizontal size threshold lines This peak should not be very high and if so one should refer to problem 2 of the trouble shooting guide An example is below JAttaGraph E Attagene Software Experimental data 7008 01 09 _Plate512_F09 fsa o
12. f DNA from your PCR reaction Cause The Primer Extension labeling reaction failed Solution Make sure that the labeling reaction protocol on your thermocycler is the same profile as indicated in the protocol Note only 1 cycle is used Solution The Primer Extension reaction was lacking a component repeat the Primer Extension protocol Solution The fluorophore conjugated oligonucleotide was stored improperly or the expiration date has passed Problem 2 Small or No peak heights for the Reporter blue peaks except for the 680 nt peak Full lenght from capillary electrophoresis but your standard red peaks look fine Cause Incomplete digestion of the Labeled DNA Solution Repeat the labeling and digestion procedure Make sure that you use the recommended incubation temperature and duration You may increase the digestion time to at least 5 hours Also you can run aliquot on gel see picture below Problem 3 Many TF s have low or zero value Note It is common for some TF s to have low corresponding values Cause Blue Threshold is not correctly adjusted Solution Adjust the blue threshold you must open the preference file under the File option Cause Corresponding TF activity is low Solution Try another cell type Solution Try another version of FACTORIAL Solution Call us for custom design 22 Problem 4 Blue and or Red peaks atypical as the capillary run progresses Cause 1 Capillary Array may be o
13. j si Size threshold lines Peak produced by Under digested Fraction size 680nt LKE ECO KOHL HE oa Y y I Ir a UV LS LS jj E F j T 5 After you have checked the under digested peak and adjusted the threshold you may extract the TF activity data 17 6 To look at the tab delimited data select the show peaks data button in far left corner of the tools menu You will get another pop up window showing the data OO OO OO OO O OO Ol O WEE Copy Peaks FsaFileName 2 Danny Software 3 new Calibration ABI files Jan 9 0812008 01 09_plateS12_E09 fsa FactorialFileName factorialName Num Size height Area Pos DS CS 116 34 1807 132 16 1999 144 28 4129 180 68 2821 186 67 1176 192 78 3017 231 5 2300 241 61 1920 266 41 2144 272 76 2214 283 47 1632 301 39 1649 317 34 1693 322 43 1824 332 43 1954 338 44 953 344 51 3372 350 58 2131 359 7 1508 364 67 1880 369 34 2140 377 14 1555 394 34 2469 408 29 1201 428 86 2174 442 68 1770 464 44 2487 481 19 1075 554 08 1217 562 5 813 578 45 1274 586 43 740 602 61 752 LLB 100 17045 4526 21500 4773 40623 4966 28685 5554 11627 5651 30583 5750 24944 6400 20683 6571 24046 6995 25283 7104 17990 7288 19926 7596 19577 7872 21265 7960 23324 8133 10932 8237 40655 8342 27197 8447 18294 8605 23447 8691 26986 8772 18824 8907 31541 9205 15485 9447 29488 9804 Relative Peak Height Specific TF Activity TF Name 1 162 1 286 2 65
14. lds the calibration files using the ctrl or shift key to select at least three files then choose open 4 Open Lookin cabat er el alle CA ai 2008 01 09_plateS12_E09 fsa l gt Calibration files My Recent Documents My Documents PL My Computer d SI File name 1 09 plateS12_FO9 fsa 2008 01 09 glate Gg Feat Open My Network Places Files of type All Fies Lei 12 7 The following window will open and allow you to double check that the correct calibration files are present after review select calibrate Then you will see the dialog box populated with the differences between Pre set and calibrated values which is shown below After review select Save Factorial Calibration is now complete and you can close this window zi Calibrate Dialog Z Documents and Settings admin O1 THERALOGIC Cry Documents and Settings Admin 01 THERaALOGIC Cry Documents and Settings Admin 01 THERaALOGIC FRALOGICSO01 0O0 FRALOGICSO0O1 O0 FRALOGICSO0O1 O0 5794066171193 51853257685975 6042 5324703067 0648569319595105 612351510644565 37479659455433 55363257959175 5356974726069 142 4665 465207 903972037005 T T0010S93205 455959906385696 MAr i e e a 69281614275159 694721873679 651377723555 F SS540835 437334 98243501523401 Note The size difference between pre set and calibrated reporter values should be no more than 3 base pairs
15. ne click and hold then move up or down B ewe C Documents and Settings Admin 01 THERALOGICSO01 000 Deskto OES BAD l Grey line is threshold for noise and can be adjusted so it includes all calibration peaks Name SREBP Size 338 4393063903915 ij pos 8z37 Height 953 Area 9168 Lha HARA WI UI Note a The red line is the size standard threshold it can be adjusted from the advanced options but is not recommended b The grey line is the TF threshold and can be adjusted by clicking and dragging up or down in the window 10 4 Leave the above reporter file window open and then select from the main frame Attagene window Calibration Calibrate factorial as shown below 4 JAttaGraph Joe File Calibration Edit Factorial Calibrate Factorial Select several files and calibrate Factoria ajta gene You are using Factorial 30A ATTAGRAFH Reader v 1 4 8 2008 Open Cal Files Calibrate Save Factorial SREBP yop LXE 5 Once you select calibrate factorial you will see a pop up window that will allow you to select which files you want to use for calibration Click on the open Cal Files button which is shown circled above 11 6 From this window you can browse and choose which calibration files that you wish to use They should be from the current run that corresponds to the data that has been generated Search for the appropriate folder that ho
16. ta button and open Excel then select paste or right click and paste To copy only TF activity click on the copy TF activity button and follow the same route as the copy all data Once you are satisfied with the data you can view the data in a radar style graph within our program Alternatively you can also do this in Excel You can close this window when done Below is an example of all data copied to Excel and radar graphs drawn of specific activity and histograms drawn of relative peak height for the 30 TF s M o 7 i Num Size height Area Pos TF zm Specific TF ES 1 108 4 67 am 4389 TGF Activity 3 2 116 67 537 mp 4499 TATA D a 131 85 145 mum 4748 TCF Apo lily ERE GES 1 144 16 523 en 4942 PPR Wii Ls ay E 5 180 59 265 2329 5524 GRE A M BE aS NF kB 7 6 men pe 16020 8620 AP 1 aei HTT HAN oy Ore St D z 192 7 192 1694 6718 ISRE le ae SETH ER 8 231 62 459 4430 6362 NF k GATA RE TTN Xbp1 10 9 241 63 am 3181 6531 FoxA M Le Mi GAN 10 266 43 541 395 6951 Xbpt NRF 1 vi NEE CRE a 11 272 42 1627 17370 7053 CRE ci AV RG SERS bes E DAN 1 A 13 12 283 39 438 4526 7240 AhrE d 14 13 301 34 2731 2788 7548 ARE ARE 15 14 317 21 up 4376 og ERE 16 15 322 33 1077 12094 7907 Oct Az 16 332 38 3421 3626 8080 HSE 19 17 338 37 1808 21184 8183 SRE 19 18 344 42 746 8489 8287 p53 20 19 350 52 792 9454 8392 BRE 2
17. ve a policy of altering analyzing the fragment analysis files if this is the case please inform the sequencing facility that only raw fsa files without previous analysis are required for this software Computer Requirements PC or Mac Windows 98 or later operating system Internet access to connect to the software Java 4 5 or greater to run the software We can also provide fragment analysis as a service at our facility near the Research Triangle Park NC This is for clients who don t have access to ABI sequencers or for clients that wish to take advantage of this part of the assay as aservice Please refer to our assay manual for instructions on this process Installation and Registration of ATTAGRAPH 1 Connect to the internet and proceed to our webpage at Attagene com 2 Click on link to connect with ATTAGRAPH software you will need to register and log into the software 3 Once you see the security pop up message you will need to select run 4 Once the program has opened the following screen shot should appear showing which FACTORIAL version you re using JAttaGraph Pm FOXO a w E Box alta gene You are using Factorial 30A ATTAGRAPH Reader v 1 4 3 2008 Tee P SREBP oer LXR oe lf this screen does not appear then reconnect to the internet and follow the instructions from above If problems persist please contact info atiagene com or by phone at 888 721 2121 ll
18. verused and need replacing 2 Running buffer is old 3 POP 7 is expired Solution Replace defective reagents or array and re run your samples Cause Instrument settings incorrect Solution Use correct settings refer to appendix A Problem 5 Extra peaks are seen in the capillary electrophoresis plot This is a normal profile eer a a 1 re a a Fees rr degen Weber beet T Le sf Er Leg elai aeii i be r Kin wem aial Wes a a 23 This profile below has extra peaks i 1 d UI 8 See UI WG e E A E E VM Lt An AL DO r aT UNL Le iT LA A TTE Cause Too much PCR product was used for the labeling reaction Solution Recheck the amount of PCR product against the mass ladder If there is too much PCR product present then use 1 20 of the reaction for labeling Problem 6 Some or all of the Reporter blue peaks from the capillary electrophoresis plot are unnamed Note Itis normal to have some un named reporter peaks this is not an issue unless most peaks are missing Cause The molecular weight standards that were added to your labeled and processed DNA sample were not the standards supplied with your kit Solution Rerun your samples but use only the supplied capillary electrophoresis standards Cause The calibration run has not been performed to help ATTAGRAPH determine where the reporter peaks migrate on your capillary electrophoresis machine Solution Run the calibration standards supplied in your kit
19. which corresponds to three integers in the table For example 364 should never go higher than 367 or lower than 361 13 8 Open another calibration file and set grey threshold to cross all calibration blue peaks then make sure there are no un named peaks and all major peaks have positive values You can open the tab delimited data to review these values by selecting the show peaks data button below 5 J ttatst aph Denny Solhware ss new Caliban ARI files Jan 9 O20 09 plate Ti 2 E fa Renom a a LA J 8 8 LA LC Copy Peaks saFileName 2 Danny Softwares new Calibration ABI Files Jan 9 0812008 01 09_plate512_G09 fFsa FactorialFileMame Factorialwame Num Size height Area Pos 109 06 1570 116 54 1271 132 23 1449 144 42 2604 180 9 1984 166 59 366 192 95 2245 231 71 1863 241 85 1525 266 67 1713 272 99 167Z 203 75 1333 301 7 1305 317 63 1431 z2 r4 1377 332 04 1653 330 84 734 Ad DO 2774 350 94 1790 360 08 1239 365 01 1573 369 01 1765 of 7 50 1279 394 72 2108 406 68 1052 429 31 1699 443 14 1458 464 89 2143 431 61 932 oot 1016 562 81 707 576 83 1127 596 51 662 602 94 6 76 611 1 1089 OO sl Oh Of E GO ba rz Eech bech OC 148517 4359 1 226 12000 4473 0 994 15312 4716 1 133 27596 4908 2 192 19572 5491 ol B479 S587 U D 4 eefoe 2684 1 755 40658 6426 1 457 16522 6495 1 191 19147 6915 1 339 19042 7020 1 307 147380 7202 1 042 15445 7506 1 020 16586 7777 1 119 162355 786 4
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