title: GSM3939074: NbBtsI_rep1; Saccharomyces cerevisiae; OTHER
library_construction_protocol: Genomic DNA was prepared using a spheroplast-based genomic DNA extraction kit (Qiagen, 10243) according to the instruction manual. Isolated DNA was treated with restriction or nicking endonucleases, left untreated (samples 1 and 2) or subjected to random fragmentation to an average size of ca. 300 bp by sonication (samples 19 and 20). EndoSeq libraries (samples 9 and 10) were prepared according to a published protocol (doi: 10.1038/nprot.2015.099).   GLOE-Seq libraries (all other samples) were prepared as follows:   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.