DNA-Protein Crosslink sequencing (DPC-seq) in RPE-1 cells treated with formaldehyde
CUT&Tag was performed as previously described84 in high salt conditions in biological triplicate. A reference, pre-treatment sample was collected 48h post-transduction. Every other day, cells were counted and replated at 500X library coverage in fresh medium with or without formaldehyde or 5-aza-dC at doses calculated to cause 50% growth inhibition (155µM and 55nM, respectively). Representative cell pellets were harvested for genomic DNA extractions, library preparation and NGS at day 2 (initialome), at day 10 (essentialome) and at day 24 (endpoint drug treatment).  Genomic DNA (gDNA) was extracted using the QIAamp DNA Blood Maxi Kit (Qiagen) as per the manufacturer's instructions.  200 μg RNA (in a total volume of 116 μl) was fragmented by adding 4 μl of 5 M NaOH and left on ice for 30 min. Fragmentation was stopped by addition of 80 μl of 1 M Tris pH 7 and cleaned up twice using the Micro Bio-Spin P-30 Gel Columns (BioRad, 7326223) according to the manufacturer's instructions. Biotinylation of 4SU-residues was performed in a total volume of 250 μl, containing 10 mM Tris-HCl pH 7.4, 1 mM EDTA and 5 mg of MTSEA biotin-XX linker (Biotium, BT90066) for 30 min at room temperature in the dark. RNA was then purified by phenol–chloroform extraction, denatured by 10 min incubation at 65 °C and added to 150 μl μMACS Streptavidin MicroBeads (Milentyl, 130-074-101). RNA was incubated with beads for 15 min at room temperature and beads were applied to a μColumn in the magnetic field of a μMACS magnetic separator. The beads were washed twice with 55°C pull-down wash buffer (100 mM Tris-HCl, pH 7.4, 10 mM EDTA, 1 M NaCl and 0.1% Tween-20). Biotinylated RNA was eluted twice by addition of 100 mM DTT and cleaned up using the RNeasy MinElute kit (Qiagen, 74204) using 1,050 μl ethanol (≥99%) per 200 μl reaction after addition of 700 μl of RLT buffer to precipitate RNA of less than 200 nucleotides. At least 30 ng of the purified 4SU-labelled RNA was then used as input for the TruSeq Stranded Total RNA kit (Illumina, 20020596) for library preparation. The libraries were amplified according to the manufacturer's instructions. The library was amplified with 10 PCR cycles and quality-control checked on the TapeStation (Agilent) using the High Sensitivity DNA kit before pooling and paired-end sequencing on the NextSeq 550 (Illumina) system. Per condition, 200,000 untreated, formaldehyde-treated or released cells were resuspended in Wash buffer (20mM HEPES pH 7.5, 600 mM NaCl, 0.5 mM Spermidine, 1x Protease inhibitor cocktail) and prepared with Concanavalin A (ConA)-coated magnetic beads (Bangs Laboratories, BP531) beads. Primary antibodies (Table 3) were incubated 1:50 overnight. Cells were washed with Dig-wash buffer (0.05% digitonin, 20 mM HEPES pH 7.5, 600 mM NaCl, 0.5 mM spermidine in nuclease-free water with a Roche Complete Protease Inhibitor EDTA-free tablet) and secondary antibody (Table 3) was added 1:50 and incubated for 2 hr at 25oC. Cells were washed three times with 300 mM NaCl Dig-wash buffer, and pA-Tn5 tagmentation was performed 1:250 in Dig-300 buffer (0.01% digitonin, 20 mM HEPES pH 7.5, 300 mM NaCl, 0.5 mM spermidine in nuclease-free water with a Roche Complete Protease Inhibitor EDTA-free tablet). Library preparation was then performed by subjecting a total of 561ug of gDNA from the reference sample and 5.84ug (across 29 and 6 PCR plates of 100ul reactions, respectively) from experimental samples to 28 PCR cycles with NEBNext Ultra II Q5 Master Mix (New England Biolabs), using custom indexed primers (Table 2) for the simultaneous amplification of the lentiviral backbone and addition of Illumina adapter sequences. PCR products were then purified by running in 1% agarose gels followed by gel extraction using the Qiaquick Gel Extraction Kit (Qiagen) as per the manufacturer's instructions.