GSM7850262: WT HAP1 cells exposed to 50 nM trabectedin, biological replicateR1; Homo sapiens; OTHER
For the Nb.BsrDI treatment experiment, genomic DNA was extracted using the Monarch genomic DNA purification kit (T3010) from untreated U2OS wildtype cells after harvesting (5 min incubation with 0.25 % trypsin). For the experiments with cells, nuclei isolation followed the GLOE-seq protocol for mammalian cells (PMID: 32320643) with the following modification. Isolation of genomic DNA from mammalian cell culture: Agarose plugs were incubated with 6 mL of proteinase K solution and shaken at 170 rpm. For the Nb.BsrDI treatment experiment, DNA library for Illumina sequencing was prepared according to GLOE-Seq protocol 'Steps y23 – 28: Denaturation and ligation of 3'-OH termini (yeast)' onwards (PMID: 32320643). For the experiments with cells, nuclei isolation and DNA library preparation for sequencing followed the GLOE-seq protocol for mammalian cells (PMID: 32320643) with the following modifications. Isolation of genomic DNA from mammalian cell culture: Agarose plugs were incubated with 6 mL of proteinase K solution and shaken at 170 rpm. Fragmentation and capture of biotinylated single-stranded DNA: DNA was sonicated with an average fragment length of 300 nucleotides with Qsonica for 5 min at 4 °C in cycles of 15 s ON and 5 s OFF with 20 % amplitude. DNA was purified twice with AMPure beads with elution in 50 μL dH2O. Streptavidin MyOne C1 dynabeads were resuspended after washing in 50 μL bind and wash buffer (2x) for a 1:1 beads to sample ratio. Second strand synthesis, end polishing and ligation of the distal adaptor: Reagents for second strand synthesis, end polishing and distal adaptor ligation were combined on ice and mixed by pipetting. For the distal adaptor either 3792-UMI or 3792-UMI.v2 (different indexes) were annealed with 3791. DNA was purified with AMPure beads and eluted in 30 μL of dH2O. qPCR: Before the final library amplification described in (Sriramachandran et al., 2020), qPCR with reaction conditions identical to the final PCR used for library amplification was done for quality control. Samples (1 μL) were diluted in deionized water (9.4 μL) and combined with 1x Q5 buffer, 0.2mM dNTPs, 0.1 μM i5 illumina indexing primer, 0.1 μM P7-short primer, 1x EvaGreen and 0.1 units of Q5 high-fidelity DNA polymerase. qPCR protocol included 120 s initial denaturation at 95 °C, 40 cycles of 15 s denaturation at 95 °C, 30 s annealing at 60 °C and 20 s extending at 72 °C, as well as 95 s final extension at 72 °C. Library amplification: The library was scaled up from 20 μL to 50 μL using entire samples. A customized P7-short primer compatible with the UMI-containing distal adaptors was used. Samples were purified with AMPure beads with a 1:1 bead to sample ratio. The DNA libraries were sequenced on an Illumina NovaSeq 6000 with a single-read protocol and the read length of 101 nucleotides (R1); additionally, 15 cycles were run on R2 to read UMI and custom indexes introduced by the oligos 3792-UMI or 3792-UMI.v2. For each sample, we deposited these R1 and R2 sequencies. Supplementary Table 1 of the manuscript (titled the same way) summarizes the reagents, enzymes, kits, oligonucleotides, and other materials used for GLOE-seq experiments.