GSM5005325: G1_setB_rep1_TrAEL-seq; Saccharomyces cerevisiae; OTHER
For yeast: Cells in ethanol (1-3x107 per plug) were pelleted in round bottom 2 ml tubes by centrifuging 30 s 20,000 g, washed once in 1 ml PFGE wash buffer (10 mM Tris HCl pH 7.5, 50 mM EDTA) and resuspended in 60 µl same with 1 µl lyticase (17 U/ µl in 10 mM KPO4 pH7, 50% glycerol, Merck >2000 U/mg L2524). Samples were heated to 50°C for 1-10 min before addition of 40 µl molten CleanCut agarose (Bio-Rad 1703594), vortexing vigorously for 5 s before pipetting in plug mould (Bio-Rad 1703713) and solidifying 15-30 min at 4°C. Each plug was transferred to a 2 ml tube containing 500 µl PFGE was buffer containing 10 µl 17 U/µl lyticase and incubated 1 hour at 37°. Solution was replaced with 500 µl PK buffer (100 mM EDTA pH 8, 0.2% sodium deoxycholate, 1% sodium N-lauroyl sarcosine, 1 mg/ml Proteinase K) and incubated overnight at 50°. Plugs were rinsed with 1 ml TE, then washed three times with 1 ml TE for 1-2 hours at room temperature with rocking, 10 mM PMSF was added to the second and third washes from 100 mM stock (Merck 93482). Plugs were then digested 1 h at 37°C with 1 µl 1000 U/ml RNase T1 (Thermo EN0541) in 200 µl TE. RNase A was not used as it binds strongly to single stranded DNA [76]. For hESC: 1x106 cells were washed once in 5 ml L buffer (10 mM Tris HCl pH 7.5, 100 mM EDTA, 20 mM NaCl) and resuspended in 60 µl L buffer in a 2 ml tube. Samples were heated to 50°C for 2-3 min before addition of 40 µl molten CleanCut agarose (Bio-Rad 1703594), vortexing vigorously for 5 s before pipetting in plug mould (Bio-Rad 1703713) and solidifying 15-30 min at 4°C. Each plug was transferred to a 2 ml tube containing 500 µl digestion buffer (10 mM Tris HCl pH 7.5, 100 mM EDTA, 20 mM NaCl, 1% sodium N-lauroyl sarcosine, 0.1 mg/ml Proteinase K) and incubated overnight at 50°. Washing as for yeast. TrAEL-seq: ½ an agarose plug of cells was equilibrated once in 100 µl 1x TdT buffer (NEB) for 30 min at room temperature, then incubated for 2 h at 37°C in 100 µl 1x TdT buffer containing 4 µl 10 mM ATP and 2 µl Terminal Transferase (NEB M0315L). Plugs were rinsed with 1 ml tris buffer (10 mM Tris HCl pH 8.0), equilibrated in 100 µl 1x T4 RNA ligase buffer (NEB) containing 40 µl 50% PEG 8000 for 1 hour at room temperature then incubated overnight at 25°C in 100 µl 1x T4 RNA ligase buffer (NEB) containing 40 µl 50% PEG 8000, 1 µl 10 pM/µl TrAEL-seq adaptor 1 ([App]NNNNNNNNAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTUGCGCAGGCCATTGGCC[BtndT]GCGCUACACTCTTTCCCTACACGACGCT) and 1 µl T4 RNA ligase 2 truncated KQ (NEB M0373L). Plugs were then rinsed with 1 ml tris buffer, transferred to 15 ml tubes and washed three times in 10 ml tris buffer with rocking at room temperature for 1-2 hours each, then washed again overnight under the same conditions. Plugs were equilibrated for 15 min with 1 ml agarase buffer (10 mM Bis-Tris-HCl, 1 mM EDTA pH 6.5), then the supernatant removed and 50 µl agarase buffer added. Plugs were melted for 20 min at 65°C, transferred for 5 min to a heating block pre-heated to 42°C, 1 µl β-agarase (NEB M0392S) was added and mixed by flicking without allowing sample to cool, and incubation continued at 42°C for 1 h. DNA was ethanol precipitated with 25 µl 10 M NH4OAc, 1 µl GlycoBlue, 330 µl of ethanol and resuspended in 10 µl 0.1xTE. 40 µl reaction mix containing 5µl Isothermal amplification buffer (NEB), 3 µl 100 mM MgSO4, 2µl 10 mM dNTPs and 1µl Bst 2 WarmStart DNA polymerase (NEB M0538S) was added and sample incubated 30 min at 65°C before precipitation with 12.5 µl 10 M NH4OAc, 1 µl GlycoBlue, 160 µl ethanol and re-dissolving pellet in 130 µl 1xTE.  END-seq: ½ an agarose plug of cells was equilibrated for 1 h at room temperature in 100µl NEBuffer 2 with 0.1mM dNTPs, then overnight at 37°C with 100 µl NEBuffer 2 with 0.1 mM dNTPs and 1 µl Klenow (NEB M0210S). Note that this is not the same blunting regimen used in END-seq [11] as Klenow performed best for SfiI-digested yeast genomic DNA in our hands. After rinsing twice with 1 ml tris buffer, plug was transferred to a 15 ml tube and washed three times for 15 min each with 10 ml tris buffer on rocker at room temperature before transfer to a new 2 ml tube. The plug was equilibrated with 100 µl CutSmart buffer containing 5 mM DTT and 1 mM dATP for 1 hour at room temperature before incubation for 2 h at 37°C in another 100 µl of the same buffer containing 1 µl Klenow exo- (NEB M0212S) and 1 µl T4 PNK (NEB M0201S). Plug was rinsed twice with 1 ml tris buffer then washed once with 10 ml of tris buffer for 15 min as above then returned to a 2 ml tube. The plug was equilibrated for 1 h at room temperature in 100 µl 1x Quick Ligation buffer (NEB B6058S) containing 2.7 µl END-seq adaptor 1 ([Phos]GATCGGAAGAGCGTCGTGTAGGGAAAGAGTGUU[BtndT]U[BtndT]UUACACTCTTTCCCTACACGACGCTCTTCCGATC∗T), then overnight at 25°C with another 100 µl of the same buffer containing  2.7 µl END-seq adaptor 1 and 1 µl high concentration T4 DNA Ligase (NEB M0202M). After rinsing twice with 1 ml tris buffer, plug was transferred to a 15 ml tube and washed three times for 1-2 h each with 10 ml tris buffer on rocker at room temperature, then again overnight.  The plug was transferred to a 1.5 ml tube and equilibrated 15 min with 1 ml agarase buffer (10 mM Bis-Tris-HCl, 1 mM EDTA pH 6.5), then the supernatant removed and 50 µl agarase buffer added to the plug. Plug was melted 20 min at 65°C then transferred for 5 min to a heating block pre-heated to 42°C, 1 µl beta-agarase (NEB M0392S) was added and mixed by flicking without allowing sample to cool, and incubation continued at 42°C for 1 h. DNA was ethanol precipitated with 25 µl 10 M NH4OAc, 1 µl GlycoBlue, 330 µl of ethanol and resuspended in 130 µl 1x TE, 15 min at 65°C. The DNA was transferred to an AFA microTUBE (Covaris 520045) and fragmented in a Covaris E220 using duty factor 10, PIP 175, Cycles 200, Temp 11, then transferred to a 1.5 ml tube containing 8 µl pre-washed Dynabeads MyOne streptavidin C1 beads (Thermo, 65001) re-suspended in 300 µl 2xTN (10 mM Tris pH 8, 2 M NaCl) along with 170 µl water (total volume 600 µl) and incubated 30 min at room temperature on a rotating wheel. Beads were washed once with 500 µl 5 mM Tris pH 8, 0.5 mM EDTA, 1 M NaCl, 5 min on wheel and once with 500 µl 0.1x TE, 5 min on wheel before re-suspension in 25 µl 0.1x TE. 3.5 μl NEBNext Ultra II End Prep buffer, 1 µl 1 ng/µl sonicated salmon sperm DNA (this is added as a carrier) and 1.5 μl NEBNext Ultra II End Prep enzyme were added and reaction incubated 30 min at room temperature and 30 min at 65°C. After cooling, 1.25 µl 10 pM/µl TrAEL-seq adaptor 2 ([Phos]GATCGGAAGAGCACACGTCTGAACTCCAGTCUUUUGACTGGAGTTCAGACGTGTGCTCTTCCGATC*T ) or END-seq adaptor 2c ([Phos]GATCGGAAGAGCTATTATTTAAATTTTAATTUGACTGGAGTTCAGACGTGTGCTCTTCCGATC*T), 0.5 µl NEBNext ligation enhancer and 15 µl NEBNext Ultra II ligation mix were added and incubated 30 min at room temperature. The reaction mix was removed and discarded and beads were rinsed with 500 µl wash buffer (5 mM Tris pH 8, 0.5 mM EDTA, 1 M NaCl) then washed twice with 1 ml wash buffer for 10 min on wheel at room temperature and once for 10 min with 1 ml 0.1x TE. Libraries were eluted from beads with 11 µl 1x TE and 1.5 µl USER enzyme (NEB) for 15 min at 37°C, then again with 10.5 µl 1x TE and 1.5 µl USER enzyme (NEB) for 15 min at 37°, and the two eluates combined. An initial test amplification was used to determine the optimal cycle number for each library. For this, 1.25 μl library was amplified in 10 µl total volume with 0.4 µl each of the NEBNext Universal and any NEBNext Index primers with 5 µl NEBNext Ultra II Q5 PCR master mix. Cycling program: 98°C 30s then 18 cycles of [98°C 10 s, 65°C 75 s], 65°C 5 min. Test PCR was cleaned with 8 µl AMPure XP beads (Beckman A63881) and eluted with 2.5 µl 0.1x TE, of which 1 µl was examines on a Bioanalyser high sensitivity DNA