ࡱ> PROO 'bjbjCC .R)e)eJVbb8dWL'$(#######$%(d#-##r r r #r #r r r Șd@r z##0'$r (j(r (r r ##,F'$(b m: Ultra II mRNAseq library preparation -This protocol uses the NEBNext Ultra II Directional RNAseq kit. Note that the PCR mix can be purchased separately as you will use more of this than is provided. -Use filter tips and RNase free water throughout. -Remember to remove AMPure beads from fridge ~15min before use. AMPure beads are much more cost effective when purchased in larger amounts, and we split into 1ml aliquots stored at 4 -Tube options-use LoBind 0.5ml tubes throughout the protocol except for the final PCR reaction, or use PCR tubes. This depends on the available magnetic racks and personal preference -Each time reagents are combined, mix well by pipetting >5 times. -When removing supernatant on magnetic rack for the final wash before elution from beads, make sure to get it all - I remove most with a P200, leave it for a minute then use a P20 with a P10 tip to get the remaining liquid -All reagents are either provided with the kit or we have dedicated stocks for library making dont use normal bench-top reagents -Before you start, you need to know what indexes your libraries will have we sequence up to 48 yeast libraries per lane, each has a different index. Coordinate with the people sharing your lane to find out which indexes are available. Preparation Mix 4l NEBNext First Strand Synthesis Reaction Buffer 1l NEBNext Random Primers 5l water final volume should be 10l Store mix on ice Poly(A) purification, fragmentation and priming This uses the NEBNext poly(A) prep module (in the fridge) -Dilute 10ng-500ng total RNA to 50l with water (150ng has been working great) -Re-suspend NEBNext Oligo dT beads by vortexing and transfer 15l to a fresh tube -Wash beads by adding 100l 2x RNA Binding Buffer, mix -Separate on magnetic rack for 2 min then discard the supernatant, remove from rack -Wash beads with another 100l 2x RNA Binding Buffer as above, remove from rack -Re-suspend beads in 50l 2x RNA binding buffer, then add the 50l to diluted RNA -Incubate 65 for 5 min in PCR machine (lid at 75), cool to 4, remove from machine -Re-suspend the beads by pipetting, incubate 5 min at RT -Place tube on magnetic rack for 2 min, then discard the supernatant -Remove the tube from the rack, re-suspend beads in 200l wash buffer -Place tube on magnetic rack for 2 min, then discard the supernatant -Wash the beads again with another 200l of wash buffer -Remove the tube from the rack, re-suspend beads in 50l Tris buffer to elute -Place the tube in the PCR machine (heated lid 90), heat to 80 for 2 minutes then cool to 25 -Remove the tube and add 50l 2x RNA binding buffer, mix, incubate 5 min at RT -Re-suspend the beads by pipetting, incubate 5 min at RT -Place tube on magnetic rack for 2 min, then discard the supernatant -Remove the tube from the rack, re-suspend beads in 200l wash buffer -Place tube on magnetic rack for 2 min, then discard the supernatant It is critical here to remove all the wash give the tubes a quick spin, return to the magnetic rack and remove the last liquid with a P10 tip -Remove the tube from the rack and re-suspend the beads in 6l of the First Strand mix from the preparation step (above) -Incubate at 94 for 15 minutes in PCR machine (heated lid 99) then IMMEDIATELY (as soon as the tube is cool enough to handle) place tube on ice for 1min. Make sure you are standing next to the PCR machine when it finishes. While this is incubating, label tubes for elution. -Quickly spin down tubes, place on magnet for 2min and transfer 5l of the supernatant to a clean tube First and second strand cDNA synthesis 1st Strand-On ice-To the fragmented RNA from the last step, add 4l NEBNext Strand Specificity Reagent 1 l NEBNext First Strand Synthesis Enzyme Mix final volume should be 10l -Incubate the sample in PCR machine (heated lid 80): 10 min at 25 15 min at 42 15 min at 70 hold at 4 2nd Strand-On ice-Immediately add the following reagents: 4l Second Strand Synthesis Reaction Buffer with dUTP Mix 2l Second Strand Synthesis Enzyme Mix 24l water final volume should be 40l Keep the tube on ice as you mix by pipetting up and down. -Incubate 1 hour at 16 in PCR machine (heated lid off) AMPure clean up Per sample, make >400l 80% ethanol fresh (use within a few hours, do not use old 80% ethanol) make from dedicated ethanol stock and nuclease free water Warm AMPure beads to RT and vortex to re-suspend completely -Add 72l AMPure beads to reaction (1.8x), pipette to mix, incubate 5 min at RT -Spin briefly to collect liquid and place on magnetic rack for 5 min until mix is clear -Remove supernatant and leave tube on rack -Add 200l 80% ethanol (do not re-suspend the beads), wait 30 seconds and remove -Perform a second wash with 200l ethanol as above -Remove residual ethanol with a double p20+P10 tip -Air dry the beads on the rack for 5 minutes (10 minutes absolute maximum) -Remove the tube from the magnetic rack, add 26.5l 0.1x TE to elute, vortex -Incubate 2 min at RT, spin briefly and place on magnetic rack until clear -Remove 25l of eluted cDNA to a new tube You may store the samples at -20 at this point if necessary End Prep of cDNA library and Adaptor Ligation End Prep-On ice-Add the following reagents: 3.5l NEBNext Ultra II End Prep Reaction Buffer (10x) 1.5l NEBNext Ultra II End Prep Enzyme Mix final volume should be 30l Incubate the samples in PCR machine (heated lid 75) 30 min at 20 30 min at 65 hold at 4 While this is incubating, defrost an aliquot of NEBNext Adaptor and make a 1:10 dilution in 10mM Tris pH7.5-8.0 Adaptor Ligation-On ice-Once machine reaches 4, remove samples and add: 1.25l Diluted NEBNext adaptor 0.5l NEBNext Ligation Enhancer 15l NEBNext Ultra II Ligation Master Mix final volume should be 46.75l -Incubate the samples in PCR machine (heated lid off) at 20 for 15 min -Add 3l of USER Enzyme to the ligation mixture from the previous step. -Mix well and incubate at 37 for 15 min with heated lid set to e"45 AMPure clean up Follow the AMPure clean up protocol as above, BUT Use 49.75l AMPure beads (1x) Elute with 23l 0.1x TE Transfer 21l eluted DNA to new tube You may store the samples at -20 at this point if necessary PCR amplification To the eluted DNA, add: 25l NEBNext Ultra II Q5 Hot Start PCR Master Mix 2l Universal PCR primer 2l Index (X) primer (each library uses a different index primer) final volume should be 50l Run PCR program with heated lid set to 99 98 30 seconds 98 10 seconds \ 65 75 seconds / x 12-15* 65 5 minutes 4 hold *Use 13 cycles for 150ng starting RNA, 15 cycles for 50ng, scale in between Reactions can be left in PCR machine overnight at 4 You may store the samples at -20 at this point if necessary AMPure clean up The samples get cleaned twice here to remove all traces of adaptor and primer (NEB say once but in my hands twice is much better) Follow the AMPure clean up protocol as above, BUT Use 45l AMPure beads (0.9x) Elute with 26l 0.1x TE Transfer 25l eluted DNA to new tube You may store the samples at -20 at this point if necessary Then, repeat the AMPure clean up protocol as above, BUT Use 22.5l AMPure beads (0.9x) Elute with 11.5l 0.1x TE Transfer 10.5l eluted DNA to a 1.5ml tube Ensure the final library tube is clearly labelled with the sample name, the date AND THE INDEX NUMBER. Store this at -20 -Quantify your library using Qubit dsDNA HS Assay Kit and load 1.5-2.5ng on the Bioanalyzer. Remember to take note of the dilution, ideally on the Bioanalyzer lane name. Quantification must also be tested using KAPA or NEB library quantification kits.      FILENAME mRNAseq Library preparation v1.6 Houseley lab  PAGE 2  $%&'LOg: j k l m < S   O i j x y ǿǷǯǷϪϢϯzrrrh h 6h h#k6h hM6h hU6h h`6h h|Y6h hPN6 hOu6h h6h h@ 6h hW76h hBU6 h 6hMh4'hcH5CJ(aJ(h#k5CJ(aJ(hBU5CJ(aJ(hf5CJ(aJ(,%& l    F b m WXgdUxgd|Y$a$gdcH   G H I d e m  $*+7UVWXY{۷|xۓ˓˓hEhf#hPNOJQJ hBUhPNh2hPNh_hh_h6h`hh_5CJaJhh`5CJaJhMwOJQJhMwhkRhBUOJQJhBU hBUhBUhUhhU5CJaJhOu h 6/8923lm?@xy()xygd5@ gd%79:?EJRUVm34kmn@AfgwyzļԸh&h3h5@ OJQJh5@ hOJQJhh2hUOJQJhUh_,h`OJQJh`h=~hPNOJQJh_hMwhPNB&')*DE^cyz01@ARST,-.pqs𸰸 hh5hf_h1Gh@Vhk~6h@Vh5@ 6hPEh@VhbOrh_h`6h_h5@ 6hUhW~h_h5@ OJQJh5@ h&h&OJQJ>?@rs=>?gd5@ '<=>?+,;<=KLȗȋȇ{ȋȋsoȏkȏh0hxhEOJQJh&OJQJhsTh&hEh&OJQJhsTOJQJhE`hE`5B*phh5B*phhh5H*hh5h& hKo5hh&5CJaJhho5CJaJhh5@ 5CJaJ h&5 hC)5(/>M[fg/0jkPQgdEgd^V`gd&`gdMwLefghjrsxy  ,-/0jklY|thth{1|h#k6OJQJh{1|h#k6 hE5hhE5CJaJh{1|h{1|hS6hSh^VOJQJh^Vh"(h;hEOJQJhEhoh0hohE`5B*phhE`5B*phhh5H*hh5 h&hE`h& h&h&'Y6789delm jk -./OPlm཰hh3Ap5CJaJ heB5 h6h!6B*OJQJphh6h!6B*phh!h~ h!h!hk`h=^heBh#kOJQJh#kh3hEh{1| hEhEh{1|hE6478cdij./lm-IJDFG|},3IJeijlmnst㪢ە㾍hheBh 2hznhzn5B*phhAih@ 6hAih!6h5|hhhph h!OJQJhh{1|h{1|5B*phh'h'5h!hh!5CJaJhh()5CJaJ4Jd=>: < \ ^ !4!!!!!gd6gd /<=>?DF 6 8 : < J Z \ !̲̽䋇{h3h4aOJQJh4ah hgx5hgxhgx5 h!hhh  h!hm;Rhm;R hBUh zh zh& h!h!hAihgxOJQJhDhgxhh h!OJQJh}h!hh$0!!! !2!4!L!N!!!!!!!""."/":"C"^"_"a"b"|"}""""""## # ###,#-#?#C#F#G#S#T#Z#[#a#c#p##ᾶ骢h:Whd\6h:Wh36 h6h:Whdx06hd\h|YOJQJh|Yh3hdx0h h 5 h@ 5 h6h66B*OJQJphh6h66B*phh6hgxh h OJQJh| hgxh 2!""+"_"z""""##)#D#R#Z#####$$+$,$$$$gd|Ygd6gd3`gd|Y$$gd3gd ##########$$"$*$+$,$$$$$$%%%%%%&%@%`%a%}%~%%%%%%%%%&&&&&&&ijį돋ijį랋whxuh|Y6OJQJhxuh|Y6h@ hgxh|Yh|YOJQJh|Y h|Y5hgxh|Y5h6 h6h66B*OJQJphh6h66B*phh66B*OJQJ^Jphh66B*phh3h:Wh|Y6h:Wh36.$%%?%@%}%~%%%%&&&&''''''''''''''gdgd6gd|Y&&C'''''''''''''''''''''''''''''''h60JmHnHu h(0Jjh(0JUh%h(hpGh/<+h/<+mHnHu h/<+h(jh(UhR/jhR/U hEh|Yh|YhMh~h '''''''gd|Y,1h. A!"#$% w2 0@P`p2( 0@P`p 0@P`p 0@P`p 0@P`p 0@P`p 0@P`p8XV~ 0@ 0@ 0@ 0@ 0@ 0@ 0@ 0@ 0@ 0@ 0@ 0@ 0@ 0@6666_HmH nH sH tH @`@ 6NormalCJ_HaJmH sH tH DA`D Default Paragraph FontRiR  Table Normal4 l4a (k (No List 4@4 Mr2Header  9r 4 @4 Mr2Footer  9r .)@. Mr2 Page NumberH"H Ko Balloon TextCJOJQJ^JaJN/1N KoBalloon Text CharCJOJQJ^JaJPK![Content_Types].xmlN0EH-J@%ǎǢ|ș$زULTB l,3;rØJB+$G]7O٭VGRU1a$N% ʣꂣKЛjVkUDRKQj/dR*SxMPsʧJ5$4vq^WCʽ D{>̳`3REB=꽻Ut Qy@֐\.X7<:+& 0h @>nƭBVqu ѡ{5kP?O&Cנ Aw0kPo۵(h[5($=CVs]mY2zw`nKDC]j%KXK 'P@$I=Y%C%gx'$!V(ekڤք'Qt!x7xbJ7 o߼W_y|nʒ;Fido/_1z/L?>o_;9:33`=—S,FĔ觑@)R8elmEv|!ո/,Ә%qh|'1:`ij.̳u'k CZ^WcK0'E8S߱sˮdΙ`K}A"NșM1I/AeހQתGF@A~eh-QR9C 5 ~d"9 0exp<^!͸~J7䒜t L䈝c\)Ic8E&]Sf~@Aw?'r3Ȱ&2@7k}̬naWJ}N1XGVh`L%Z`=`VKb*X=z%"sI<&n| .qc:?7/N<Z*`]u-]e|aѸ¾|mH{m3CԚ .ÕnAr)[;-ݑ$$`:Ʊ>NVl%kv:Ns _OuCX=mO4m's߸d|0n;pt2e}:zOrgI( 'B='8\L`"Ǚ 4F+8JI$rՑVLvVxNN";fVYx-,JfV<+k>hP!aLfh:HHX WQXt,:JU{,Z BpB)sֻڙӇiE4(=U\.O. +x"aMB[F7x"ytѫиK-zz>F>75eo5C9Z%c7ܼ%6M2ˊ 9B" N "1(IzZ~>Yr]H+9pd\4n(Kg\V$=]B,lוDA=eX)Ly5ot e㈮bW3gp : j$/g*QjZTa!e9#i5*j5ö fE`514g{7vnO(^ ,j~V9;kvv"adV݊oTAn7jah+y^@ARhW.GMuO "/e5[s󿬅`Z'WfPt~f}kA'0z|>ܙ|Uw{@՘tAm'`4T֠2j ۣhvWwA9 ZNU+Awvhv36V`^PK! ѐ'theme/theme/_rels/themeManager.xml.relsM 0wooӺ&݈Э5 6?$Q ,.aic21h:qm@RN;d`o7gK(M&$R(.1r'JЊT8V"AȻHu}|$b{P8g/]QAsم(#L[PK-![Content_Types].xmlPK-!֧6 0_rels/.relsPK-!kytheme/theme/themeManager.xmlPK-!g theme/theme/theme1.xmlPK-! ѐ' theme/theme/_rels/themeManager.xml.relsPK] "R HHUUUX LY!#&' "#%'J!$''!$&( 3IPRX!8@0(  B S  ? pxEMJLMOPRSUVux/27FJLMOPRSUV33333&'lmjxGGcc9U^ c R R **  DDFFbbeeii>?<>SUn  U,IEIJJMMV&'lmjxGGcc9U^ c R R **  DDFFbbeeii>?<>SUn  U,IEIJ~ 1G7at @ Ef_2w}L(OM:Wmpog.if 5@ \! #$%\'"(t)(*/<+D}+R/dx0Mr23$h6m":{;l>; BDADpGcHgH^ImQm;RkR7T@V^VY|YzYMZ[@)[s,[o[d\F^b^_E`k`QycpefgDi^kxomznKo3ApbOrMktRuuOuxu;[xz!{./|{1|5|"~W~Mh~k~x~Au[#kfL<&07(&AiMw'gx_6+0sv z~()7bR`&S|BU=~n 2Sa W7}N(LUx_< )hpPN!=^h_,h0eBsf#,Uo4asT ,AZ &# DFL* AC)Y"PE-d;JL@? ? ? ? (@ D@UnknownG.[x Times New Roman5Symbol3. .[x Arial7..{$ Calibri9. . Segoe UIC.,.{$ Calibri LightA$BCambria Math"qhW]<66!24;;3qHP ?72!xx  Prepare 0jhousele Jon Houseley Oh+'0  < H T`hpx Prepare 0 jhousele Normal.dotmJon Houseley140Microsoft Office Word@@R{Z4@d ՜.+,0 hp  University of Edinburgh6;  Prepare 0 Title  !"#$%&'()+,-./0123456789:;<=>@ABCDEFHIJKLMNQRoot Entry FЋdS1Table*(WordDocument.RSummaryInformation(?DocumentSummaryInformation8GCompObjr  F Microsoft Word 97-2003 Document MSWordDocWord.Document.89q