邢唷��>� NQ���KLM����������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������欹�O ��鴗bjbjU経� .z7讕e7讕e�*�H���������@Q0����������8�l9l�� t��(�������������$g"�%d��������������� ddd�������d��ddd�����P 漁9�����D�d�� 0� d�%�d�%dd�%����d�������04���� �������������������������������������������������������������������������%���������� �: 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 � 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 � plug 2ml tube Equilibrate plug once in 100祃 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癈  Remove the all agarase buffer, add 50 祃 agarase buffer to plug and melt 20 min at 65� Shift tubes to 42� for 5 min, then add 1ul �-agarase, flick, incubate 1 hour at 42� Be quick during addition  do not let the tube cool down Add 25�l 10M NH4OAc, 1�l glycoblue and 330�l 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 10祃 0.1xTE, >10 min at RT For mammalian samples 15 min at 65� is better but make sure it doesn抰 go dry Add master mix containing 29祃 water 5祃 Isothermal amp buffer (NEB) 3祃 100mM MgSO4 2祃 dNTPs (10mM) 1祃 Bst 2 warmstart pol (NEB) Pipette to mix and re-suspend pellet Heat 30min at 65� Add 12.5祃 10M NH4OAc, 1祃 glycoblue and 160祃 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 130祃 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 11篊 Treatment time 50s Sample volume 130祃 After use turn everything off, empty water and dry Wash 8祃 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 300祃 2xTN (10 mM Tris pH 8, 2 M NaCl) per sample, split into 300祃 aliquots in 1.5ml tubes and add 170祃 water to each Add 130祃 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 500祃 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 500祃 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 祃 0.1x TE Store overnight at 4( or carry on directly Add 3.5�l NEBNext Ultra II end repair buffer 1�l 1ng/ul salmon sperm DNA 1.5�l NEBNext Ultra II end repair enzyme Pipette to mix and incubate 30 min at 20� (room temperature) then 30 min at 65� Let cool then spin 10s at 1000rpm � Add 1.25�l new ENDseq adaptor 2 (from annealed 10uM stock) 0.5�l ligation enhancer 15�l NEBNext Ultra II ligation mix Pipette to mix and incubate 30 min at 20� (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 11祃 1x TE + 1.5�l USER enzyme Incubate 15 min at 37� This elutes the DNA from the beads Collect beads using magnet, transfer 12�l 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 10.5�l 1x TE + 1.5�l USER enzyme Incubate 15 min at 37� This elutes any remaining DNA from the beads Collect beads using magnet, add 12祃 supernatant to the 12祃 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 1.25�l library 0.4�l NEBNext any index oligo 0.4�l NEBNext universal oligo 2.95ul water 5�l NEBNext Ultra II PCR mix 98� 30s 98� 10s \ 65� 75s / x 18 cycles 65� 5 min 4� hold � Clean with 8�l AMPure beads, elute in 2.5�l 0.1x TE You can elute up to 2.0�l from these but it is easier just to put the eluate and bead mix on the magnet and pipette 1�l 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抰 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抰 seem to cause big problems in TrAEL-seq. Final library amplification: In PCR tubes, mix 21�l library 2�l NEBNext index oligo 2�l NEBNext universal oligo 25�l NEBNext Ultra II PCR mix 98� 30s 98� 10s \ 65� 75s / x calculated cycles 65� 5 min 4� 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 40�l AMPure beads, elute with 26�l 0.1xTE for 25�l yield Clean again with 20�l AMPure beads, elute with 11�l 0.1xTE for 10.5�l yield Run 1�l directly on Bioanalyser and use 0.2�l 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: 20祃 100uM ENDseq Adaptor 2 in 200ul 1x T4 DNA ligase buffer Incubate in heating block at 95癈 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\]�����     - � � � � ( ) R o p �   K T j � � � � � � � � $ 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哸坅歛渁篴糰綼繿 bb3bHbIbJbUbVbWb\bqbrb嘼攂昩躡輇cQcUcVc刢瞔d蜩蜣谚蚶蚬蜩蜩颡烌栩钁噾钁噝pfXfzph/dB*OJQJ^Jphh/dB*^Jphh蘨�B*^Jphh蘨�h蘨�B*^Jphh��B*^Jphh��h��B*^Jphhg1B*H*^Jphhg1hg1B*H*^Jph hg1hg1!h�hg1B*OJQJ^Jphha[Zha[ZB*^Jphha[ZB*^Jphhg1B*^Jphhg1hg1B*^Jph"sbb嘼輇Qc刢d$d^d燿甦痙赿鱠,e-elexe膃鱡7f8fBf�������������������������gd�� �Ggd恦�gd蘨� 勑�0齘勑`�0齡da[Zgd�d#d$d]d^dodudvd焏燿琩璬踕鰀鱠e+elesetewexe裡鰁鱡f6f7f8fBf攆蜩蜩蜥蜩谥鲜孪窒敬Т殝殎磥t乸i謉\Xhg1hg1hg16� ha[Z6� h焏Th��h��h��h��B*^Jphh��B*^Jphhg1h��B*H*^Jphhg1h��B*^Jphh恦�h恦�B*^Jphh恦�B*^Jphh��h恦�h恦�6� h蘨�6� h焏Th�h恦� h恦�h蘨� h�h蘨�h蘨�B*^Jphh蘨�h蘨�B*^Jph"BfCfbf宖靎鴉%g+g2gCgVgjgkglgmg遟)heh噃砲i{ipt畉痶眛���������������������������gd蘨�gdr�gd�攆檉踗鉬鴉2gCgjgkglgmgng遟鄃)h*hehfh噃坔峢巋環砲磆縣裩鄅iii{i|i璱tptqt畉痶豇痿祠遛责势势几犉羝湌幤幤巹靹靷靫tph�h�jh�0JUUjh/d0JUhf�jhf�0JUhr�jhr�0JUhAD8jhAD80JUhF�jhF�0JUh��jh��0JUjh蘨�0JU h焏Th� h焏Th蘨�h/dh蘨�ha[Zhg1h��(y of T4 DNA polymerase in the End repair mix so it doesn抰 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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