GSM3939107: WT_RAF_rep1; Saccharomyces cerevisiae; OTHER
Ca. 6×10^9 cells (300 OD600) were collected by centrifugation (2,000×g, 5 min), washed in 25 ml of 10 mM Tris-HCl, pH 8.5, resuspended in 4 ml of Y1 buffer (1 M sorbitol, 100 mM EDTA, pH 8.0, 14 mM ß-mercaptoethanol) and spheroplasted by addition of 400 µl of Zymolyase 20T (AMS Biotechnology, 10 mg/ml). Spheroplasts were harvested by centrifugation (2,000×g for 5 min), washed once with 4 ml of Y1 buffer without ß-mercaptoethanol, resuspended in 4.75 ml of lysis buffer (50 mM Tris-HCl, pH 8.0, 50 mM EDTA) and lysed by incubation at 37° C by with 250 µl of 20% SDS. After centrifugation at 5,000×g for 10 min, the supernatant was transferred to a fresh 50 ml tube, 1.666 ml of 5 M potassium acetate were added, and tubes were incubated on ice for 45 min. The suspension was cleared of debris by two rounds of centrifugation (17,000×g for 15 min), and DNA was precipitated by addition of 19.5 ml of 100% ethanol. After centrifugation at 8,000×g for 15 min, the pellet was washed twice with 70% (v/v) ethanol and distributed into two 2 ml microcentrifuge tubes. After air-drying, the DNA was resuspended in 245 µl of TE by incubation overnight at 4° C. After addition of 5 µl of 5 M NaCl, samples were treated with 2.5 µl of RNase A (Sigma-Aldrich, 10109169001) for 1 h at 37° C. DNA was again precipitated by addition of 1.5 ml of 100% ethanol and centrifugation at 21,100×g for 10 min. Pellets were rinsed once with 1 ml of 70% (v/v) ethanol, air-dried, and resuspended overnight at 4° C by gentle rocking in 250 µl of 10 mM Tris-HCl pH 8.5. The DNA was then purified using AMPure beads and eluted in 125 µl of 10 mM Tris-HCl, pH 8.5. Genomic DNA (2.5 µg) was incubated at 95° C for 10 min, followed by incubation on slushy ice for 5 min to denature the DNA. A ligation mix was set up by addition of 6.5 µl of 10× T4 DNA ligase buffer, 3.55 µl proximal adaptor (40 µM in 60 mM NaCl), 19.5 µl of 50% (w/v) PEG 8000, 3 µl of high-concentration T4 DNA ligase (NEB) and water up to a total volume of 65 µl. The reaction was incubated at 25° C for 1 h, followed by 2 h at 22° C. The reaction mix was supplemented with 35 µl of 5 M NaCl and 35 µl of water and bound to an equivalent of 100 µl AMPure beads. The beads were incubated at room temperature for 5 min and rinsed twice with 500 µl of 70% (v/v) ethanol, followed by elution of the DNA with 103 µl of water and incubation at room temperature for 5 min. This eluate (100 µl) was subjected to sonication to a fragment size of 200 nt, using a Bioruptor Pico (4 cycles, 30 s on/30 s off). The sonicated DNA was then transferred to a microfuge tube containing 20 µl Streptavidin MyOne C1 Dynabeads (ThermoFisher Scientific, prewashed once with 1 ml 10 mM Tris-HCl pH 8.5, 2 M NaCl) and incubated for 15 min at room temperature with rotation. The beads were washed on a rotating wheel at room temperature: twice with 50 µl 1× SSC buffer each for 5 min at room temperature, once with 25 µl 20 mM NaOH for 10 min and briefly with 100 µl water. The DNA was eluted in 16 µl of water by incubation at 95° C for 5 to 10 min. For second-strand synthesis, the eluted DNA (14.85 µl) was combined with 15 µl Phusion FLASH mix (ThermoFisher Scientific) and 0.15 µl 100 µM oligonucleotide 3790, and incubated at 95° C for 2 min, 60° C for 3 min and 72° C for 2 min. The resulting double-stranded DNA was purified with AMPure beads and eluted in 19 µl water. This eluate (17 µl) was A-tailed with 2.3 µl End Prep Reaction Buffer and 1 µl End Prep Enzyme Mix (NEBNext® Ultra™ II DNA Library Prep kit, NEB) for 20° C for 30 min, followed by inactivation at 65° C for 30 min. Distal adaptor (2.25 µl, 40 µM in 60 mM NaCl) was ligated to the A-tailed DNA in the presence of 13.5 µl Ligation Master Mix, 0.45 µl Ligation Enhancer (NEBNext® Ultra™ II DNA Library Prep kit, NEB) and 8.8 µl water at 20° C for 20 min. The ligated DNA was cleaned up twice with AMPure beads, before being eluted in 20 µl water. This DNA (7.25 µl) was mixed with 5 µl 5× Q5 reaction buffer, 2.5 µl 1 µM Illumina P5 primer, 2.5 µl 1 µM Illumina P7 primer, 0.5 µl 10 mM dNTPs, 0.25 µl Q5 DNA polymerase (NEB) and 7 µl water and amplified by PCR, using the following programme: 95° C for 2 min, 8× (95° C for 15 s, 60° C for 30 s, 72° C for 20 s). The amplified DNA was cleaned up twice with AMPure beads, before being eluted in 25 µl water and sequenced following the guidelines supplied by Illumina.   Oligonucleotides:   proximal adaptor: 3898/3899;   distal adaptor: 3791/3792;   3898: CTACACGACGCTCTTCCGATCTNNNNN*N-NH2 (*: phosphorothioate bond, IDT code *; NH2: 3'-amino modification, IDT code /3AmMO/);   3899: PO4-AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTAGATCTCGTTTT-Bio (PO4: 5'-phosphorylation, IDT code /5Phos/; T-Bio: 3'-biotin-dT, IDT code /3BiodT/);   3790: CGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT;   3791: GACTGGAGTTCAGACGTGTGCTCTTCCGATCT;   3792: GATCGGAAGAGCACACGTCTGAACTCCAGTC;   P5: AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT;   P7: CAAGCAGAAGACGGCATACGAGAT(X)6GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT.