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Barts Cancer Institute Queen Mary University of London

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Namespace:Barts Cancer Institute Queen Mary University of London

# characteristics.description[].text
Protocols: Metastatic colorectal cancer tissue was provided by the Barts Cancer Tissue Bank. We accessed archived frozen tissue samples and collected fresh tissue samples from the Royal London Hospital. Fresh tissue samples were flash frozen in a dry ice/ethanol bath and stored at -80°C. 10x Genomics Multiome technology was used to generate paired snRNA- and snATAC-seq data from the same cell. Nuclei were isolated using a method based upon the salty EZ-10 V2 method (dx.doi.org/10.17504/protocols.io.buxnnxme). Frozen patient tissue was cut in a sterile dish on dry ice into a rice sized section and the remaining tissue was stored at -80°C. Frozen tissue was then transferred into 300 mL Salty-Ez10 Lysis Buffer (10 mM Tris-HCl pH 7.4, 146 mM NaCl, 1 mM CaCl2, 21 mM MgCl2, 0.03% Tween-20, 1% BSA, 10% EZ lysis buffer [Sigma, NUC101-1KT], 1 U/mL RNase inhibitor [Merck, 3335399001], 1 mM DTT, nuclease free water) in a 1.5 mL tube and the sample was homogenised by stroking 15x with a douncer (Fisher Scientific, 13236679), keeping the sample on wet ice. 700 mL Salty-Ez10 lysis buffer was added and the sample was pipette mixed using a wide-bore pipette tip. The sample was then incubated for 3 minutes on wet ice, and was pipette mixed with wide-bore tips twice during the incubation. The nuclei suspension was then passed through a 70 µm strainer (Fisher Scientific, 15346248) into a fresh 1.5 mL tube. Nuclei were then centrifuged at 500 RCF for 5 minutes at 4°C, before discarding the supernatant. Nuclei were resuspended in 500 mL WRB2 buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% Tween-20, 1% BSA, 0.01% digitonin [Thermo Fisher, BN2006], 1 U/mL RNase inhibitor [Merck, 3335399001], 1 mM DTT, nuclease free water), gently pipetting using wide-bore tips. Nuclei suspension was then passed through a 40 mm strainer (Sigma, BAH136800040) into a fresh 1.5 mL tube. Nuclei were then centrifuged at 500 RCF for 5 minutes at 4°C before discarding the supernatant. Nuclei were then washed by re-suspending in 500 mL WRB2 buffer using wide-bore pipette tips and the centrifugation step was repeated. Nuclei were then re-suspended using a wide-bore pipette tip in 10-40 μL 1X nuclei buffer (10x Genomics, 2000153) depending on the size of the pellet. Nuclei were counted by diluting 2 μL of the nuclei suspension 10-fold with WRB2 buffer. 10 µL of diluted nuclei were then added to 10 μL dead cell stain (2% Ethidium homodimer-1 [ThermoFisher, E1169], 30% glycerol, nuclease free water) and counted on a Countess II FL Automated Cell Counter (ThermoFisher, AMQAF1000). Downstream processing was performed immediately using Chromium Next GEM Single Cell Multiome ATAC + Gene Expression Reagent kits (10x Genomics, 1000285).