GSM8682760: Spheroplasts incubated with TMP without 5s of UV treatment; Saccharomyces cerevisiae; OTHER
Directly after TMP photobinding, the cells were fixed in 95 ml 70% -20 °C ethanol and placed at -20°C overnight. Cells were then harvested by centrifugation at 10,000 g for 10 minutes, resuspended in 500 µl of 50 mM sodium citrate (Sigma, 567446), pH 7.4, and incubated at room temp for 10 minutes. Centrifugation and wash steps were repeated once more, followed by a 20-minute Zymolyase treatment as detailed above. Spheroplasts were harvested by centrifugation and resuspended in 100 µl of lysis buffer (300 mM NaCl (Fisher, BP358), 50 mM EDTA (Fisher, AM9260G), 2% SDS (Sigma, 71725), and 5 % (v/v) RNase A (Thermo , 12091021) for ~2 hours and then vortexed for 2 minutes. 10 µl of Proteinase K (NEB, P8107S) was added, and cells were incubated for 2 hours at 65 °C.  Zero torsion samples were harvested 10,000 g at 4°C, the supernatant was removed, and they were resuspended in 100 µl of Lysis buffer containing 10 µl Proteinase K. The zero torsion samples were incubated at 65°C overnight to reverse formaldehyde crosslinks. After lysis, genomic DNA was extracted from all samples via 2X phenol-chloroform (Sigma, 77617) extraction followed by isopropanol (Sigma, 59304) precipitation. Extracted genomic DNA was sonicated to ~400 bp fragments using appropriate settings on a Covaris E200 sonicator.  Following sonication, DNA was treated with 5 µl RNase A and purified using a Zymoclean DNA  Clean and Concentrator-5 kit according to the manufacturer's protocol using a 7:1 binding buffer:sample ratio. After quantification using a Biotum HS DNA quantification kit, 250 ng of DNA was diluted into 86 µl of 10 mM Tris-Cl pH 8.5, incubated at 98°C for 15 minutes, and then directly moved to ice for 2 minutes. DNA was then digested with 4 µl of ExoI (NEB, M0293) in 1X ExoI reaction buffer at 37°C for 1 hour. This digestion process was repeated by incubating at 98°C for 1 hour and then adding an additional 4 µl of ExoI followed by another incubation at 37°C for 1 hour. Digested samples were purified using a Zymo DNA Clean and Concentrator-5 kit using a 7:1 binding buffer: sample ratio. DNA was again quantified using the Biotum HS DNA assay. Illumina adaptors were ligated to the digested samples using the NEBNext Ultra II DNA library prep kit for Illumina (NEB, E7645) at half the standard total reaction volume of the manufacturer's protocol. Adapter ligated DNA was purified with 2X Ampure beads (Beckman Coulter) and eluted in 40 µl of 10 mM Tris-Cl pH 8.5.  DNA was stored at -20 C until further processing.  To locate psoralen interstrand crosslinks, DNA from the previous step was incubated with 5 µl of lambda exonuclease (NEB, M0262) in the provided buffer in a 50 µl total reaction volume at 37°C for 1 hour, purified with 1.8X Ampure beads, and eluted with 30 µl of 10 mM Tris pH 8.5. Lambda-digested DNA was then subjected to 10 rounds of primer extension with Q5 DNA polymerase (NEB, M0491), using 10 nM of the primer (ID): 5'-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATC*T on a thermocycler (98°C for 10s à 10 rounds (98°C, 10s à 65°C, 2 min)à 65°C, 5min à 95°C 1min à 4°C). Products were purified using 1.8x Ampure beads and eluted in 40 µl 10mM Tris pH 8.5. Primer extension products were ribotailed using 1.4 µl Terminal Transferase (NEB, M0315) and 0.4 mM rGTP (Roche, 11277057001) in Terminal Transferase buffer supplemented with 0.25 mM CoCl2 by incubating at 37°C for 30 minutes followed by incubation at 75°C for 20 min. Ribotailed products were purified with 1.8X Ampure beads, eluted with 30 µl 10mM Tris pH 8.5. Purified ribotailed products were ligated with 0.33 µM annealed adapter oligos (IDT) (Top: 5'p-AGA TCG GAA GAG CGT CGT GTA GGG AAA GAG TGT-3', Bottom: 5' ACA CTC TTT CCC TAC ACG ACG CTC TTC CGA TCT CCC -3') using 1.2 µl T4 DNA ligase (NEB, M0202S) in the provided buffer for 1 hour at 16 °C, and subsequently purified with 1.8X Ampure beads and eluted in 20 µl 10mM Tris pH 8.5. Finally, products were indexed and amplified using NEBNEXT Multiplex Oligos for Illumina set 1 (NEB, E7600) following the manufacturers protocol with a total of 12 PCR cycles. The PCR products were purified using 0.8X Ampure beads and eluted in TE (Invitrogen). Samples were pooled at equimolar concentrations and empty adapters were removed from the pool.  Pooled libraries were sequenced on a NextSeq 2000 (Illumina) using a NextSeq 2K P2 100 bp kit (Illumina) in paired-end mode with a 63 bp Read_1 and 59 bp Read_2.