ࡱ> 796 %bjbj 0&JJ.... :$.^^^^^^^^,/ "v9^^^^^^^Q^^^^V@\PH<Տ`" g0TW#W#\\><^^^^^^W#^^^^^^^^^J :   Annealing TruSeq adaptors Adaptor Annealing Protocol Creates 100ul of 15uM indexed adaptor duplex Resuspend adapter oligonucleotides at a concentration of 100 M in standard 1mM EDTA solution Mix 15ul of 100uM TruSeq indexed adaptor with 15u1 of 100uM universal adaptor Add 70ul of annealing buffer Anneal oligos an a thermal cycler with the following program: a. 2 min at 95  C 140 cycles of 20 sec at 95 C (decrease temp 0.5  C every cycle) Hold at 4C Freeze annealed adaptors in 10ul aliquots at -80C 1X TruSeq Annealing buffer: 10 mM Tris-HCl, pH 7.9 0.1 mM EDTA, pH 8.0 50 mM NaCl For 50ml: 0.5ml 1M Tris-HCl, pH 7.9 0.01 ml 500mM EDTA 0.5ml 5M NaCl 49ml H20 Optimal index pools: Some sequencing experiments require the use of fewer than 12 index sequences in a lane with a high cluster density. In such cases, a careful selection of indexes is required to ensure optimum cluster discrimination by having different bases at each cycle of the index read. Illumina recommends the following sets of indexes for low-level pooling experiments. Pool of 2 samples: Index #6 GCCAAT Index #12 CTTGTA Pool of 3 samples: Index #4 TGACCA Index #6 GCCAAT Index #12 CTTGTA Pool of 6 samples: Index #2 CGATGT Index #4 TGACCA Index #5 ACAGTG Index #6 GCCAAT Index #7 CAGATC Index #12 CTTGTA Pool of 12: 2, 4, 5, 6, 7, 12, 13, 14, 15, 16, 18, 19 OR 1, 3, 8, 9, 10, 11, 20, 21, 22, 23, 25, 27 Order all primers HPLC purified Order individual indexes at 50nM scale Universal adaptor at 2uM scale will give 150ul of 100uM oligo, enough for ~10 indices **INDEXED ADAPTORS MUST BE ORDERED WITH 5' PHOSPHATE ** For universal adapter, place phosphorothioate bond between final C and T Adapter Name Sequence Index TS001 TruSeq Adapter, Index 1 5 GATCGGAAGAGCACACGTCTGAACTCCAGTCACATCACGATCTCGTATGCCGTCTTCTGCTTG ATCACG TS002 TruSeq Adapter, Index 2 5 GATCGGAAGAGCACACGTCTGAACTCCAGTCACCGATGTATCTCGTATGCCGTCTTCTGCTTG CGATGT TS003 TruSeq Adapter, Index 3 5 GATCGGAAGAGCACACGTCTGAACTCCAGTCACTTAGGCATCTCGTATGCCGTCTTCTGCTTG TTAGGC TS004 TruSeq Adapter, Index 4 5 GATCGGAAGAGCACACGTCTGAACTCCAGTCACTGACCAATCTCGTATGCCGTCTTCTGCTTG TGACCA TS005 TruSeq Adapter, Index 5 5 GATCGGAAGAGCACACGTCTGAACTCCAGTCACACAGTGATCTCGTATGCCGTCTTCTGCTTG ACAGTG TS006 TruSeq Adapter, Index 6 5 GATCGGAAGAGCACACGTCTGAACTCCAGTCACGCCAATATCTCGTATGCCGTCTTCTGCTTG GCCAAT TS007 TruSeq Adapter, Index 7 5 GATCGGAAGAGCACACGTCTGAACTCCAGTCACCAGATCATCTCGTATGCCGTCTTCTGCTTG CAGATC TS008 TruSeq Adapter, Index 8 5 GATCGGAAGAGCACACGTCTGAACTCCAGTCACACTTGAATCTCGTATGCCGTCTTCTGCTTG ACTTGA TS009 TruSeq Adapter, Index 9 5 GATCGGAAGAGCACACGTCTGAACTCCAGTCACGATCAGATCTCGTATGCCGTCTTCTGCTTG GATCAG TS010 TruSeq Adapter, Index 10 5 GATCGGAAGAGCACACGTCTGAACTCCAGTCACTAGCTTATCTCGTATGCCGTCTTCTGCTTG TAGCTT TS011 TruSeq Adapter, Index 11 5 GATCGGAAGAGCACACGTCTGAACTCCAGTCACGGCTACATCTCGTATGCCGTCTTCTGCTTG GGCTAC TS012 TruSeq Adapter, Index 12 5 GATCGGAAGAGCACACGTCTGAACTCCAGTCACCTTGTAATCTCGTATGCCGTCTTCTGCTTG CTTGTA TS013 TruSeq Adapter, Index 13 5 GATCGGAAGAGCACACGTCTGAACTCCAGTCACAGTCAACAATCTCGTATGCCGTCTTCTGCTTG AGTCAA TS014 TruSeq Adapter, Index 14 5 GATCGGAAGAGCACACGTCTGAACTCCAGTCACAGTTCCGTATCTCGTATGCCGTCTTCTGCTTG AGTTCC TS015 TruSeq Adapter, Index 15 5 GATCGGAAGAGCACACGTCTGAACTCCAGTCACATGTCAGAATCTCGTATGCCGTCTTCTGCTTG ATGTCA TS016 TruSeq Adapter, Index 16 5 GATCGGAAGAGCACACGTCTGAACTCCAGTCACCCGTCCCGATCTCGTATGCCGTCTTCTGCTTG CCGTCC TS018 TruSeq Adapter, Index 18 5 GATCGGAAGAGCACACGTCTGAACTCCAGTCACGTCCGCACATCTCGTATGCCGTCTTCTGCTTG GTCCGC TS019 TruSeq Adapter, Index 19 5 GATCGGAAGAGCACACGTCTGAACTCCAGTCACGTGAAACGATCTCGTATGCCGTCTTCTGCTTG GTGAAA TS020 TruSeq Adapter, Index 20 5 GATCGGAAGAGCACACGTCTGAACTCCAGTCACGTGGCCTTATCTCGTATGCCGTCTTCTGCTTG GTGGCC TS021 TruSeq Adapter, Index 21 5 GATCGGAAGAGCACACGTCTGAACTCCAGTCACGTTTCGGAATCTCGTATGCCGTCTTCTGCTTG GTTTCG TS022 TruSeq Adapter, Index 22 5 GATCGGAAGAGCACACGTCTGAACTCCAGTCACCGTACGTAATCTCGTATGCCGTCTTCTGCTTG CGTACG TS023 TruSeq Adapter, Index 23 5 GATCGGAAGAGCACACGTCTGAACTCCAGTCACGAGTGGATATCTCGTATGCCGTCTTCTGCTTG GAGTGG 4n$ (  ; < $%%% hhUhhyhy5hyhX2hhz?hhX2h546n $ & ` b     & > S _ ` ^gdz?^gd & Fgdgd` j -/CTef{)*6gdygdgd6`c3i?wGQ%f=gdgdy=~k$$$%"%&%C%m%%%%%%%gdgdTS025 TruSeq Adapter, Index 25 5 GATCGGAAGAGCACACGTCTGAACTCCAGTCACACTGATATATCTCGTATGCCGTCTTCTGCTTG ACTGAT TS027 TruSeq Adapter, Index 27 5 GATCGGAAGAGCACACGTCTGAACTCCAGTCACATTCCTTTATCTCGTATGCCGTCTTCTGCTTG ACTGAT TS-UA AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATC*T PCR amplification primers MC703.TruSeq.p1 caagcagaagacggcatacgaga MC704.TruSeq.p2 aatgatacggcgaccaccgagatc OR p1 CAAGCAGAAGACGGCATACGAGAT p2 AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGA .:p/ =!"#$% 666666666vvvvvvvvv666666>6666666666666666666666666666666666666666666666666hH6666666666666666666666666666666666666666666666666666666666666666666666666662 0@P`p2( 0@P`p 0@P`p 0@P`p 0@P`p 0@P`p 0@P`p8XV~_HmH nH sH tH @`@ NormalCJ_HaJmH sH tH DA D Default Paragraph FontRiR  Table Normal4 l4a (k (No List T`T & List Paragraph ^m$OJPJQJ^JR^`R # 0 Normal (Web)dd[$\$CJOJQJaJPK!pO[Content_Types].xmlj0Eжr(΢]yl#!MB;.n̨̽\A1&ҫ QWKvUbOX#&1`RT9<l#$>r `С-;c=1g'}ʅ$I1Ê9cY<;*v7'aE\h>=,*8;*4?±ԉoAߤ>82*<")QHxK |]Zz)ӁMSm@\&>!7;ɱʋ3װ1OC5VD Xa?p S4[NS28;Y[꫙,T1|n;+/ʕj\\,E:! t4.T̡ e1 }; [z^pl@ok0e g@GGHPXNT,مde|*YdT\Y䀰+(T7$ow2缂#G֛ʥ?q NK-/M,WgxFV/FQⷶO&ecx\QLW@H!+{[|{!KAi `cm2iU|Y+ ި [[vxrNE3pmR =Y04,!&0+WC܃@oOS2'Sٮ05$ɤ]pm3Ft GɄ-!y"ӉV . `עv,O.%вKasSƭvMz`3{9+e@eՔLy7W_XtlPK! ѐ'theme/theme/_rels/themeManager.xml.relsM 0wooӺ&݈Э5 6?$Q ,.aic21h:qm@RN;d`o7gK(M&$R(.1r'JЊT8V"AȻHu}|$b{P8g/]QAsم(#L[PK-!pO[Content_Types].xmlPK-!֧6 -_rels/.relsPK-!kytheme/theme/themeManager.xmlPK-!!Z!theme/theme/theme1.xmlPK-! ѐ'( theme/theme/_rels/themeManager.xml.relsPK]#  &% ` 6=% en8> )+,4BDVXY]s{EK}M S  # W ] + 1   l r CIZ`<Sf~::hf XM}g<[\e^`o()^`.pL^p`L.@ ^@ `.^`.L^`L.^`.^`.PL^P`L.^`.^`.pp^p`.@ @ ^@ `.^`.^`.^`.^`.PP^P`.^`o(. ^`hH. pL^p`LhH. @ ^@ `hH. ^`hH. 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