Homo sapiens
Protocols: RPE1 WT or CSB-/- cells were seeded in 6 well plate at 160000 cells/well in 10% FBS containing media in technical triplicate. The following day, to synchronize cells in G1, media was removed, cells were washed once with PBS, and 1% FBS-containing media was added. 24h later, cells were treated with 1.75mM formaldehyde to induce DPCs. After 1h, formaldehyde-containing media was removed and cells were washed twice with PBS. Cells were either harvested immediately or allowed to recover for 6h. Cells were harvested by scraping in 150µl 2% SDS, 20 mM Tris/HCl pH 7.5, transferred to 1.5ml tubes, snap frozen in liquid nitrogen and stored at -80°C.  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).  For KCl/SDS precipitation, samples were thawed at 55°C for 5 min (1200 rpm shaking) and sonicated using Covaris® Focused ultrasonicator E220evo in 130µl tubes (microTUBE AFA Fiber Pre-Slit Snap-Cap 6x16mm; 1x cycle, 120 sec). Next, samples were transferred to 1.5ml tubes and 270µl 2 %SDS, 20 mM Tris/HCl pH 7.5 was added to a final total volume of 400µl. DNA extraction was performed on 10% of the total lysate using the GeneJET Genomic DNA Purification Kit (Thermo Scientific, 10410450) and considered as Input. For KCl/SDS precipitation of the remaining lysate, 400µl of 200mM KCl, 20mM Tris/HCl pH 7.5 was added to each sample, followed by incubation on ice for 5min and full speed centrifugation at 4°C (5 min). Supernatants (soluble DNA) were transferred to a new tube for DNA quantification. Pellets (containing protein and protein+crosslinked DNA) were washed three times according to the following steps: addition of 400µl 200 mM KCl, 20 mM Tris–HCl (pH 7.5), incubation at 55°C for 5 min (1200 rpm shaking), incubation on ice (5 min), full speed centrifugation 4°C (5min). Next, pellets were resuspended in 400 µl 200 mM KCl, 20 mM Tris–HCl (pH 7.5) containing Proteinase K (0.2mg/ml) and incubated at 55°C for 45 min (800 rpm shaking) for protein digestion. 10µl UltraPure BSA (Thermo Scientific, AM2616, 50 mg/ml) was added followed by cooling on ice for 5 min, followed by centrifugation maximum speed at 4oC for 5 min. Next, supernatants containing crosslinked DNA were collected. Soluble and crosslinked DNA samples were treated with 0.2mg/ml DNAse-free RNAse A (Sigma-Aldrich) for 30min at 37°C. DNA concentrations were determined using Qubit™ dsDNA HS Assay Kit (Thermo Scientific) according to the manufacturer's instructions. Relative DPC amounts between samples were calculated using the ratio between crosslinked DNA and total DNA (crosslinked plus soluble DNA) as a surrogate for DPC persistence. 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.  50ng of DNA was concentrated via ethanol precipitation with 300mM sodium acetate, 1µL glycogen and 2.5x ethanol and resuspending in 20µL of nuclease-free water. DNA was run on 1% agarose gels cast in 1.5mm Mini-PROTEAN casettes (Bio-Rad), stained with SYBR-Gold and the DNA fragment smear of 400-1000bp was dissected from the gel. Gel slices were immersed in 500µL gel-extraction buffer (10mM Tris pH 8.0, 1mM EDTA, 0.02% SDS) and rotated at 4C overnight. The gel slices and buffer were loaded into Spin-X columns (Costar, CLS8160) and span at max speed for 10 minutes at 4C. The eluted DNA containing buffer was ethanol precipitated as before and resuspended in 50µL nuclease free water. DNA was then subjected to library preparation via the NEBNext Ultra-II DNA library prep kit (NEB, E7645L) using a 1 in 10 adapter dilution and 7 PCR cycles.
isolate
not applicable
organism
Homo sapiens
organism
Homo sapiens
cell_line
hTERT RPE-1
collection date
not collected
scientific_name
Homo sapiens
common name
human
organism part
eye
cell_type
retinal cell
genotype
CSB-/-
geographic location (country and/or sea)
not collected