GSM4015617
GSM4015617: 3xCUP1_24hr_1_REC-seq; Saccharomyces cerevisiae; OTHER
Percoll gradients (1-2 per sample depending on final OD) were formed by vortexing 1.16ml Percoll (Sigma P1644) with 42 µl 5 M NaCl, 98 µl water in 2 ml tubes and centrifuging 15 min at 15,000 g, 4 °C. Cells were defrosted, washed with 25 ml cold PBSE and re-suspended in a minimal amount of PBSE and layered on the pre-formed gradients. Gradients were centrifuged for 4 min at 2,000 g, then the upper phase and brown layer of cell debris removed and discarded. 1 ml PBSE was added, mixed by inversion and centrifuged 1 min at 2,000 g to pellet the cells, which were then re-suspended in 1ml PBSE (re-uniting samples split across two gradients). 25 µl Streptavidin magnetic beads were added (Miltenyi Biotech 1010007) and cells incubated for 15 min on a wheel at room temperature. Meanwhile, 1 LS column per sample (Miltenyi Biotech 1050236) was equilibrated with cold PBSE in 4 °C room. Cells were loaded on columns and allowed to flow through under gravity, washed with 1 ml cold PBSE and eluted with 1ml PBSE using plunger. Cells were re-loaded on the same columns after re-equilibration with ~500 µl PBSE, washed and re-eluted, and this process repeated for a total of three successive purifications. 50 µl cells were set aside for quality control, while 1 μl 10% Triton X-100 was added to the remainder which were then pelleted by centrifugation and frozen or processed directly for DNA extraction. Cell pellets were re-suspended in 50 µl 0.34 U/ml lyticase (Sigma L4025) in 1.2 M sorbitol, 50 mM EDTA, 10 mM DTT and incubated at 37° C for 45 min. After centrifugation at 1,000 g for 5 min, cells were gently re-suspended in 80 µl of 0.3% SDS, 50 mM EDTA, 250 μg/ml Proteinase K (Roche 3115801) and incubated at 65° C for 30 min. 32 µl 5 M KOAc was added after cooling to room temperature, samples were mixed by flicking, and then chilled on ice for 30 min. After 10 min centrifugation at 20,000 g, the supernatant was extracted into a new tube using a cut tip, 125 µl phenol:chloroform pH 8 was added and samples were mixed on a wheel for 30 min. Samples were centrifuged for 5 min at 20,000 g, the upper phase was extracted using cut tips, and precipitated with 250 μl ethanol. Pellets were washed with 70% ethanol, air-dried and left overnight at 4° C to dissolve in 20 µl TE. All reagents from NEB except where noted otherwise. 20 μl genomic DNA isolated from aged cells as above was diluted to 155 μl final volume containing 1x CutSMART and 0.75 μl RNase T1 (Fermentas), and incubated 15 min at room temperature before splitting into 3x 49 μl and 1x 3μl aliquots (the latter was diluted to 16 μl for total DNA isolation). The 49 μl aliquots were digested with 1 μl EagI-HF, PvuI-HF or 0.5μl PvuII-HF for 4 hours at 37 °C, 0.5 μl additional enzyme was added to the PvuII digest every hour. For mutant analysis, PvuII was replaced with SmaI and this digestion was performed at 25 °C. 6 μl 10 mM ATP, 1 μl 10x CutSmart, 1 μl Exonuclease V and 1 μl Exonuclease I were added to each digest and incubated over night at 37 °C before extraction with phenol chloroform and ethanol precipitation in the presence of 1 µl GlycoBlue (Life Technologies). DNA pellets were dissolved in 45 μl 0.1x TE, then 6 μl 10x CutSmart, 6 μl 10 mM ATP, 1 μl Exonuclease V, 1 μl Exonuclease I and 1 μl same restriction enzyme as previous day were added and samples again incubated over night at 37 °C before extraction with phenol chloroform and ethanol precipitation. For SmaI digestions, the enzyme and CutSmart were added first and digestion performed for 4 hours at 25 °C before addition of other components and shift to 37 °C for overnight incubation. Pellets were dissolved in 33 μl 0.1x TE and the 3 aliquots combined, precipitated again with ethanol and pellets dissolved in 16 μl 0.1x TE. Exonuclease treated and total DNA samples were incubated at 37 °C for 45 min with 2 μl NEBNext DNA fragmentase  and 2 μl fragmentase buffer, then 5 μl 0.5 M EDTA was added followed by 25 μl water and samples were purified using 50 μl AMPure beads and eluted with 25.5 μl 0.1x TE. 3.5 μl NEBNext Ultra II end prep buffer and 1.5 μl  NEBNext Ultra II end prep enzyme were mixed in and samples incubated 30 min at 20 °C then 30 min at 65 °C. After addition of 1.25 μl 1:25 diluted NEBNext adaptor, 0.5 μl ligation enhancer and 15 μl NEBNext Ultra II ligation mix samples were incubated 15 min at 20 °C, then 1.5 μl USER enzyme was added and incubated 15 min at 37 °C. Samples were cleaned with 44 μl AMPure beads, eluted with 30 μl 0.1x TE, then cleaned again with 27 μl AMPure beads and eluted with 22.5 μl 0.1x TE. 1.25 μl library was amplified with 0.4 μl each NEBNext index and universal primers and 5μl NEBNext Ultra II PCR mix in 10 μl total volume using recommended cycling conditions with 8 cycles for total samples, 16 cycles for 48 hour aged samples and 18 cycles for 24 hour samples. These test amplifications were cleaned with 9 μl AMPure beads, eluted with 1.5 μl 0.1x TE and 1 μl run on a Bioanalyzer to determine the optimal cycle number for the final amplification. Library yield was aimed to be 2 pM, the test amplification should be equivalent to a 1:4 dilution of the final library. The remaining library (21 μl) was then amplified in a 50 μl reaction containing 2 μl each NEBNext index and universal primers and 25 μl NEBNext Ultra II PCR mix using the calculated cycle number, cleaned with 45 μl AMPure beads, eluted with 25 μl 0.1x TE, then cleaned again with 22.5 μl AMPure beads and eluted with 10.5 μl 0.1x TE.
GEO
GSM4015617
GEO Accession
GSM4015617