ÐÏࡱá>þÿ NQþÿÿÿKLMÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿì¥ÁO ø¿øtbjbjU½U½ .z7׎e7׎eÒ*ïHÿÿÿÿÿÿ·ÌÌ@Q0���ÿÿÿÿ•••8Íl9l•ó t¥¥(ÍÍͨ¨¨²´´´´´´$g"¶%dØÕ�¨¨¨¨¨Ø��ÍÍÛ­ ddd¨œ�Í�Ͳd¨²dddÍÿÿÿÿP �O9ØÿÿÿÿDˆdžà 0ó d�%Ìd�%dd�%�‚¨¨d¨¨¨¨¨ØØ04¨¨¨ó ¨¨¨¨ÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿ�%¨¨¨¨¨¨¨¨¨Ì ×: TrAEL-seq Reagents and consumables are detailed at the end of the protocol Input material Start with agarose plugs containing 1-4x10^7 yeast cells that were ethanol fixed at harvest or 1-2x10^6 mammalian cells (see protocol for Agarose Embedding), you need ½ of a plug per sample. Stopping points This protocol is very long but can be done in bits, and we have stopped at many points without problems. To our knowledge all the following stopping points are fine: You can pause indefinitely at any point where plugs are in tris buffer, store at 4° You can pause indefinitely when DNA is in solution in TE, store at 4°. You can pause indefinitely when DNA solutions have ethanol added for precipitation, is a pellet in the 70% ethanol, or the pellet is re-suspending in TE. Store at 4°. You can pause indefinitely when DNA is bound to magnetic beads once beads are in bead wash or TE, store at 4°. Vortex well to re-suspend when you re-start. Once you start the library preps using the NEBNext Ultra II kit, you must continue until the beads are in the wash solution, but then you can store at 4°. After eluting the DNA from the magnetic beads with USER, store the libraries in the freezer until ready to amplify. Similarly, amplified libraries before or after purification are stored in the freezer. Protocol Transfer ½ plug 2ml tube Equilibrate plug once in 100µl 1x TdT buffer, 30 min at room temperature (RT) Exchange for 100ul containing: 10ul 10x Terminal transferase buffer (NEB) 4ul 10mM ATP , 1ul Terminal transferase (NEB) 84ul water Incubate 2-3 hrs at 37 Discard the solution and rinse plug with 1ml tris buffer Then equilibrate plug 1 hr at RT with: 10ul 10x T4 RNA ligase buffer (NEB) 40ul 50% PEG 8000 50ul water Exchange for 100ul containing: 10ul 10x T4 RNA ligase buffer 40ul 50% PEG 8000 1ul App TrAEL adaptor 1 48ul water 1ul T4 RNA ligase 2 KQ Mix all components except ligase in a 1.5ml tube, vortex vigorously, then add ligase and pipette repeatedly with a P200 cut tip to mix it really well Incubate overnight at 25deg [NOT LONGER!!!] Discard reaction mix, rinse plug 1x 1ml tris buffer, transfer to 15ml tube During the day wash 4x with 10ml tris buffer at room temperature on rocker Leave overnight on rocker with 10ml tris buffer Put plug in normal 1.5ml tube and equilibrate 15min or more with 1ml agarase buffer Meanwhile, heat a block to 65° and another to 42°C  Remove the all agarase buffer, add 50 µl agarase buffer to plug and melt 20 min at 65° Shift tubes to 42° for 5 min, then add 1ul ²-agarase, flick, incubate 1 hour at 42° Be quick during addition  do not let the tube cool down Add 25µl 10M NH4OAc, 1µl glycoblue and 330µl of ethanol, vortex vigorously (particularly for mammalian samples) Chill 15 min on ice then spin 15 min top speed 4° Wash with 70% ethanol, dry 5 min, resuspend in 10µl 0.1xTE, >10 min at RT For mammalian samples 15 min at 65° is better but make sure it doesn’t go dry Add master mix containing 29µl water 5µl Isothermal amp buffer (NEB) 3µl 100mM MgSO4 2µl dNTPs (10mM) 1µl Bst 2 warmstart pol (NEB) Pipette to mix and re-suspend pellet Heat 30min at 65° Add 12.5µl 10M NH4OAc, 1µl glycoblue and 160µl of ethanol, vortex well Chill 15 min on ice then spin 15 min top speed 4° Wash with 70% ethanol, dry 5 min, resuspend in 130µl 1xTE 15min at 65° Can proceed directly or store at 4° Transfer to Covaris tubes (AFA Snap-cap microTUBE) Machine use: Turn on machine Turn off and restart! Turn on computer Turn on chiller Fill water tank to 6L Fill in booking sheet Protocol (Cris in JHGroup folder) Duty factor 10% Peak Incident Power 175W Cycles 200 Temp 11ºC Treatment time 50s Sample volume 130µl After use turn everything off, empty water and dry Wash 8µl Dynabeads MyOne streptavidin C1 beads per sample twice with 0.5ml bead prep buffer (5mM Tris pH8, 0.5mM EDTA, 1M NaCl, 0.05% Tween), 3 min on wheel per wash [you can wash beads for up to 4 samples together] Resuspend beads in 300µl 2xTN (10 mM Tris pH 8, 2 M NaCl) per sample, split into 300µl aliquots in 1.5ml tubes and add 170µl water to each Add 130µl sonicated DNA to beads Incubate on wheel 30 min at RT Spin tubes up to 1000rpm before putting on magnet to collect liquid Wash beads 1x 500µl 5mM Tris ph8, 0.5mM EDTA, 1M NaCl, 5 min on wheel Spin tubes up to 1000rpm before putting on magnet to collect liquid Wash beads 1x with 500µl 0.1x TE, 5min on wheel Spin tubes up to 1000rpm before putting on magnet to collect liquid Carefully remove the wash solution so as not to leave any drops and re-suspend beads immediately in 25 µl 0.1x TE Store overnight at 4( or carry on directly Add 3.5¼l NEBNext Ultra II end repair buffer 1µl 1ng/ul salmon sperm DNA 1.5¼l NEBNext Ultra II end repair enzyme Pipette to mix and incubate 30 min at 20º (room temperature) then 30 min at 65º Let cool then spin 10s at 1000rpm   Add 1.25¼l new ENDseq adaptor 2 (from annealed 10uM stock) 0.5¼l ligation enhancer 15¼l NEBNext Ultra II ligation mix Pipette to mix and incubate 30 min at 20º (room temperature) Remove reaction mix using magnet, rinse beads 500ul wash buffer (5mM Tris ph8, 0.5mM EDTA, 1M NaCl). REMEMBER your libraries are on the beads! Wash beads 2x 1ml wash buffer (5mM Tris ph8, 0.5mM EDTA, 1M NaCl) 10 min on wheel each Spin tubes up to 1000rpm before putting on magnet to collect liquid Wash beads 1x with 1ml 0.1x TE, 10min on wheel Spin tubes up to 1000rpm before putting on magnet to collect liquid Carefully remove the wash solution so as not to leave any drops and re-suspend beads immediately in 11µl 1x TE + 1.5¼l USER enzyme Incubate 15 min at 37º This elutes the DNA from the beads Collect beads using magnet, transfer 12µl supernatant to a 1.5ml LoBind tube using an orange LoBind tip The supernatant contains the library but we do another round of elution to get anything left. This is the first step in which the very low concentration DNA is unprotected, hence the LoBind tubes and tips Resuspend beads in 10.5µl 1x TE + 1.5¼l USER enzyme Incubate 15 min at 37º This elutes any remaining DNA from the beads Collect beads using magnet, add 12µl supernatant to the 12µl from the first elution using an orange LoBind tip Store samples at -20 if necessary We then perform a test amplification to work out how many cycles we need to use for the real library. In PCR tubes, mix 1.25¼l library 0.4¼l NEBNext any index oligo 0.4¼l NEBNext universal oligo 2.95ul water 5¼l NEBNext Ultra II PCR mix 98º 30s 98º 10s \ 65º 75s / x 18 cycles 65º 5 min 4º hold   Clean with 8¼l AMPure beads, elute in 2.5¼l 0.1x TE You can elute up to 2.0¼l from these but it is easier just to put the eluate and bead mix on the magnet and pipette 1µl straight onto the Bioanalyzer lane Run on Bioanalyzer Use this to calculate the cycle number for the final amplification  note: because of the various dilutions, assume that the final library would be 4x more concentrated than the test library, so reduce the number of cycles by 2 before further adjustments. Try to aim for a final library concentration of 1.5-4 nM. Adaptor dimers are often seen in these test reactions, be sure to exclude from the quantification. You should quantify across the region from 300-1,000 bp, which should exclude the adaptor peaks. However, it is not uncommon to get weird profiles like these:  Don’t worry – your libraries should be fine, but quantification is not so accurate. Err on the side of slightly over amplifying the library (so only reduce the number of cycles by 1 rather than 2 before adjusting the concentration). Library over-amplification doesn’t seem to cause big problems in TrAEL-seq. Final library amplification: In PCR tubes, mix 21¼l library 2¼l NEBNext index oligo 2¼l NEBNext universal oligo 25¼l NEBNext Ultra II PCR mix 98º 30s 98º 10s \ 65º 75s / x calculated cycles 65º 5 min 4º hold Transfer libraries to 1.5 ml tubes for final purification (purification seems to be better in 1.5 ml tubes than in PCR tubes) Clean with 40¼l AMPure beads, elute with 26¼l 0.1xTE for 25¼l yield Clean again with 20¼l AMPure beads, elute with 11¼l 0.1xTE for 10.5¼l yield Run 1µl directly on Bioanalyser and use 0.2µl for KAPA quantification [see separate protocol] It is not unusual to have some residual adaptor in the library. If so, dilute to 25ul and perform another 2 rounds of AMPure bead purification at 0.8x. Reagents Adaptors TrAEL-seq adaptor 1: [Phos]NNNNNNNNAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTU GCGCAGGCCATTGGCC [BtndT] GCGCUACACTCTTTCCCTACACGAC GCT Purchase PAGE purified from Sigma-Genosys or other supplier 5’ App added using the 5’ adenylation kit (see separate protocol for this) TrAEL-seq adaptor to (aka: ENDseq adaptor 2): [Phos]GATCGGAAGAGCACACGTCTGAACTCCAGTCUUUUGACTGGAGTTCAGACGTGTGCTCTTCCGATC*T Purchase PAGE purified from Sigma-Genosys or other supplier Annealing: 20µl 100uM ENDseq Adaptor 2 in 200ul 1x T4 DNA ligase buffer Incubate in heating block at 95°C 5 min then remove block from the heat and leave to cool to room temperature Reagents and consumables Terminal Transferase (NEB M0315S) 10mM ATP (NEB) aliquot on receipt and store at -80°, keep in use aliquot at -30° for no more than 1 month    M\]Š�¸ÔÕ     - … – » Ë ( ) R o p ú   K T j ‡ ˆ › œ ¨ ¶ ¸ þ $ Q R ! 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Use home-made 50% PEG – the kit does not come with enough  Care! Not T4 DNA ligase buffer!  Home made, but same as NEB agarase buffer  Make sure these really reach temperature before use! Give them at least 20 min  With our current filter tips it is very hard to remove material from Covaris tubes – use a normal yellow tip instead  This is included to buffer the voracious activit\a†aˆašaœaºa¼a¾aÀa bb3bHbIbJbUbVbWb\bqbrb‡b”b•b¦b§bÜbÝbcQcUcVc„c²cdòèòÞÑèòÀò¹òèòèòªžòèò葇‘葇zpfXfzph/dB*OJQJ^Jphh/dB*^JphhÌi¥B*^JphhÌi¥hÌi¥B*^Jphh“�B*^Jphh“�h“�B*^Jphhg1B*H*^Jphhg1hg1B*H*^Jph hg1hg1!h™hg1B*OJQJ^Jphha[Zha[ZB*^Jphha[ZB*^Jphhg1B*^Jphhg1hg1B*^Jph"sbb‡b§bÝbQc„cd$d^d d¢d®d¯dÚd÷d,e-elexe¢eÄe÷e7f8fBfúúúúíííèúúßúúúúúúúÚúúúúúúgd“� ÆGgd�v·gdÌi¥ „Є0ý^„Ð`„0ýgda[Zgd£d#d$d]d^dodudvdŸd d¡d¢d£d¬d­dÛdöd÷de+elesetewexe¢eÑeöe÷ef6f7f8fBf”fòèòèòáòèÚÖÏÊÂÏÖϾ´§´š‹š�´�t�piÖd\Xhg1hg1hg16� ha[Z6� hŸdTh“�h“�h“�h“�B*^Jphh“�B*^Jphhg1h“�B*H*^Jphhg1h“�B*^Jphh�v·h�v·B*^Jphh�v·B*^Jphhþ÷h�v·h�v·6� hÌi¥6� hŸdTh£h�v· h�v·hÌi¥ h™hÌi¥hÌi¥B*^JphhÌi¥hÌi¥B*^Jph"BfCfbfŒf£fìføf%g+g2gCgVgjgkglgmg¢gßg)heh‡h³hi{ipt®t¯t±túúúúúúúúúúúúúúõðîîîîîîîîîììgdÌi¥gdr×gd£”f™fÛfãføf2gCgjgkglgmgng¢g£gßgàg)h*hehfh‡hˆh�hŽh­h³h´h¿hÑhàhiii{i|i­itptqt®t¯tüøôøðôìôåÞÔðÊÆÊÆ¼¸®ª ÆôÆœ’ŽÆŽÆŽ„ì„ì‚ìxtph©h£jh£0JUUjh/d0JUhfÿjhfÿ0JUhr×jhr×0JUhAD8jhAD80JUhFíjhFí0JUhþ÷jhþ÷0JUjhÌi¥0JU hŸdTh£ hŸdThÌi¥h/dhÌi¥ha[Zhg1h“�(y of T4 DNA polymerase in the End repair mix so it doesn’t degrade rare DNA fragments. It may not be necessary.  Note – do NOT use the ENDseq 2c adaptor – it will not work!      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