title: GSM8498522: CRAMP1-KO + GFP1-CRAMP1, 20 μM etoposide 1 hour, CC-seq, Rep2; Homo sapiens; OTHER
library_construction_protocol: Ethanol fixed cell pellets were lysed with 2.4 ml 1× STE buffer (2% SDS, 0.5 M Tris pH 8.1, 10 mM EDTA, 0.05% bromophenol blue), for 10 min at 65 °C, before cooling to room temperature. Extractions were performed with 1.25 ml Phenol-Chloroform-isoamyl alcohol (25:24:1; Sigma) to remove bulk free protein, then DNA in the aqueous/interphase was precipitated by addition of 3.3ml ethanol, followed by centrifugation at 10,000g for 2 min. DNA pellets were washed once with 70% ethanol, before resuspension in 1 mL 1x TE buffer pH 7.5 overnight at 4 °C. Samples were fully resuspended, prior to sonication to an average fragment size of 300–400 bp with Covaris (duty cycle: 10%, intensity/peak power incidence: 75 W, cycles/burst: 200, time: 21 min). A sample of 12 uL was removed and incubated at 37˚C for 45 min with 3 uL of RNAse A (1 mg/ml). DNA shearing was checked by 1% agarose gel electrophoresis, and DNA was quantified in technical triplicate with the Qubit (ThermoFisher) and High Sensitivity reagents. Next, S.cerevisisae DNA spike was added to 950 uL of sample (Yeast:Human DNA weight ratio of 1:100), then the total volume was made up to 1 mL with 1x TE (pH 7.5). Triton-X100, N-Lauroylsarcosine sodium salt and NaCl were then added to complete the binding buffer (final concentrations: 0.3 M NaCl, 0.2% Triton-X100, 0.1% N-Lauroylsarcosine sodium salt). Each sample was divided over two QIAquick silica fibre membrane spin columns (QIAGEN). Under these conditions, protein, but not free nucleic acids, bind to the silica membrane, leading to selective retention of any protein-DNA CCs. Columns were washed six times with 600 µL of TEN (10 mM Tris pH 7.5, 1 mM EDTA, 0.3 M NaCl) per 1 min wash to remove any residual non-CC DNA fragments, prior to elution twice with 125 µL TES (10 mM Tris pH 7.5, 1 mM EDTA, 0.5% SDS). Eluted products were pooled to 500 µL in TES and incubated with 1 mg/mL Proteinase K (Sigma) for 60 min at 50 °C, prior to overnight ethanol precipitation at −20 °C with 1.41 mL ethanol, 0.2 mg/mL glycogen and 200 mM NaOAc. The DNA-glycogen precipitate was pelleted by centrifugation at 20,000 × g for 1 h at 4 °C, washed once with 1.5 mL 70% ethanol, and air-dried for 10 min at room temperature. DNA was solubilised in 25 µL 10 mM Tris-HCl pH 7.5, and subjected to end repair and adapter ligation with NEBNext Ultra II DNA Library Preparation kit (NEB), according to manufacturer's instructions, except all steps were conducted at half-scale and a custom P7 adapter was used. After ligation of the P7 adapter to the (non-CC) sonicated end, TE was added to a final volume of 90 uL. DNA was isolated with AMPure XP beads (Beckman Coulter) according to manufacturer's instructions (beads:input of 78:90 uL) and eluted in 85 µL water. Next, 10 uL of 10X Cutsmart buffer and 5 uL Shrimp Alkaline Phosphatase (rSAP; NEB) were added before incubation for 30 min at 37˚C. DNA was isolated again with AMPure XP beads (Beckman Coulter) (beads:input of 83:100 uL) and eluted in 50 µL 10 mM Tris-HCl. Samples were diluted twofold with 50 µL TDP2 reaction buffer (100 mM TrisOAc, 100 mM NaOAc, 2 mM MgOAc, 2 mM DTT, 200 µg/ml BSA) and incubated with 3 µL of 10 µM recombinant zebrafish TDP2 catalytic domain (Addgene plasmid #200512) for 1 h at 37 °C, to non-nucleolytically repair the Top2cc DNA end. DNA was isolated again with AMPure XP beads (beads:input of 83:103) and eluted in 26 µL 10 mM Tris-HCl. Next, a second round of adapter ligation was conducted using a custom adapter dp-P5 without prior end repair, to prevent dephosphorylation of DNA 3′-phosphate termini which may be generated by TDP2 activity on 3′-CCs. After ligation of the dp-P5 adapter to the Top2-cleaved end, sample volume was adjusted to 90 uL with Tris-HCl, DNA was isolated with AMPure XP beads (beads:input of 78:90) and eluted in 15 µL 10 mM Tris-HCl. This was used as template for the NEBNext Ultra II PCR step using universal primer (P5 end) and indexed primers for multiplexing (P7 end), according to manufacturer's instructions (12–15 cycles). PCR reactions were diluted with 50 µL 10 mM Tris-HCl, DNA was isolated with AMPure XP beads (beads:input of 83:100) and eluted in 30 µL 1 mM Tris-HCl pH 8.1, 100 uM EDTA. Sample DNA molarities were quantified using the Bioanalyzer or Tapestation (Agilent).