ࡱ> NQKLMO tbjbjUU .z7׎e7׎e*H@Q08l9l t($g"%d dddddddP O9Dd 0 d%d%dd%d04 % : 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 100l 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 42C  Remove the all agarase buffer, add 50 l 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 25l 10M NH4OAc, 1l glycoblue and 330l 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 10l 0.1xTE, >10 min at RT For mammalian samples 15 min at 65 is better but make sure it doesnt go dry Add master mix containing 29l water 5l Isothermal amp buffer (NEB) 3l 100mM MgSO4 2l dNTPs (10mM) 1l Bst 2 warmstart pol (NEB) Pipette to mix and re-suspend pellet Heat 30min at 65 Add 12.5l 10M NH4OAc, 1l glycoblue and 160l 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 130l 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 11C Treatment time 50s Sample volume 130l After use turn everything off, empty water and dry Wash 8l 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 300l 2xTN (10 mM Tris pH 8, 2 M NaCl) per sample, split into 300l aliquots in 1.5ml tubes and add 170l water to each Add 130l 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 500l 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 500l 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 l 0.1x TE Store overnight at 4( or carry on directly Add 3.5l NEBNext Ultra II end repair buffer 1l 1ng/ul salmon sperm DNA 1.5l 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 1.25l new ENDseq adaptor 2 (from annealed 10uM stock) 0.5l ligation enhancer 15l 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 11l 1x TE + 1.5l USER enzyme Incubate 15 min at 37 This elutes the DNA from the beads Collect beads using magnet, transfer 12l 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 10.5l 1x TE + 1.5l USER enzyme Incubate 15 min at 37 This elutes any remaining DNA from the beads Collect beads using magnet, add 12l supernatant to the 12l 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 1.25l library 0.4l NEBNext any index oligo 0.4l NEBNext universal oligo 2.95ul water 5l NEBNext Ultra II PCR mix 98 30s 98 10s \ 65 75s / x 18 cycles 65 5 min 4 hold Clean with 8l AMPure beads, elute in 2.5l 0.1x TE You can elute up to 2.0l from these but it is easier just to put the eluate and bead mix on the magnet and pipette 1l 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:  Dont 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 doesnt seem to cause big problems in TrAEL-seq. Final library amplification: In PCR tubes, mix 21l library 2l NEBNext index oligo 2l NEBNext universal oligo 25l 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 40l AMPure beads, elute with 26l 0.1xTE for 25l yield Clean again with 20l AMPure beads, elute with 11l 0.1xTE for 10.5l yield Run 1l directly on Bioanalyser and use 0.2l 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: 20l 100uM ENDseq Adaptor 2 in 200ul 1x T4 DNA ligase buffer Incubate in heating block at 95C 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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