Powerplex 21 system

Bladder cancer cell lines (T24, UMUC3, RT4, J82) were purchased from the American Type Culture Collection (Manassas, VA, USA). Cells were grown in RPMI-1640 with L-glutamine (Thermo Fisher Scientific, Paisley, UK) supplemented with 10% foetal calf serum (Sigma-Aldrich, Dorset, UK). Cell lines were authenticated using the Promega Powerplex 21 system (Chilworth, UK) and tested for mycoplasma at the Molecular Biology Core Facility, CRUK Manchester Institute. Cells were seeded in 75-cm² flasks to obtain 60% confluency after 48 h under normoxia. After 24 h in normoxia_, media were changed and cells cultured in parallel in normoxia and hypoxia (0.2% and 1% O₂) for a further 24 h. Hypoxia was obtained using the Whitley H35 Hypoxystation (Don Whitley Scientific Limited, Bingley, UK). Experiments were carried out in biological triplicate.

A498, 786-O, and Caki1 were purchased from CLS Cell Lines Service (Eppelheim, Germany). A498 cells were cultivated in EMEM (Lonza) supplemented with 10% FBS (Merck) and 2 mM l-glutamine (Lonza). 786-O cells were cultivated in RPMI 1640 medium (Lonza) supplemented with 10% FBS and 2 mM l-glutamine. Cell lines were routinely tested for mycoplasma infection using a PCR-based test (Venor® GeM Classic, Minerva Biolabs GmbH), and authentication of cell lines was performed using the PowerPlex® 21 System (Promega) according to the manufacturer’s protocol.

Sampling was conducted by applying a wet-dry swabbing technique using viscose swabs (Forensic Swab L, Sarstedt, Nümbrecht, Germany). Upon collection, wet and dry swabs were returned to their respective tube bases, which contained a ventilation membrane for self-drying, and placed into a labelled envelope for storage. Air-dried swab tips were excised and placed into tubes (wet/dry swabs constituted one sample) and stored at −20 °C until the commencement of DNA analysis. DNA was extracted using the DNA IQ system (Promega, Madison, WI, USA) to a final volume of 60 μL and quantified with Quantifiler^® Trio DNA Quantifiler Kit on an ABIPRISM^® 7500 (Life Technologies, Carlsbad, CA, USA) and interpreted using HID software. Amplification was carried out using the PowerPlex^®21 system (Promega, Madison, WI, USA) for 30 cycles using 0.5 ng of template DNA, or, if the sample concentration was below 0.033 ng/μL,15 μL of the sample was used. Amplified product detection and sizing was performed on a 3500 xL Genetic Analyser (Life Technologies, Carlsbad, CA, USA) with an injection voltage of 1.2 kV and an injection time of 24 s. GeneMapper^® ID-X software (v1.5, Life Technologies, Carlsbad, CA, USA) was used for genotyping, with an analytical threshold of 175 RFU, as per laboratory protocol.

HT1080, SKUT1, sNF96.2, 93T449, SW684, SW872 and SW982 were purchased from the American Type Culture Collection (ATCC, Teddington, Middlesex, UK), and cultured according to their recommendations. Cell lines and culture conditions are summarized in Supplementary Table 1. Cell lines were authenticated by the Promega Powerplex 21 System and underwent mycoplasma screening (Molecular Biology Core Facility, CRUK Manchester Institute, UK).

The cultured OCI-AML3 cells were dispensed in RPMI/PBS-medium and replicates of single, 2, 5 and 10 cells were subsequently separated by micromanipulation under a dissection microscope, using a fine glass pipette. The 25 and 50 cell sample replicates were created using serial dilutions. The aliquots of 1, 2, 5, 10, 25 and 50 cells were manipulated into 200 μL PCR tubes in a total volume of 4 μL PBS and prepared for WGA (REPLI-g Single Cell Kit, Qiagen, Hilden, Germany). Genomic characterization of WGA DNA included 21 short tandem repeat loci (STR, PowerPlex 21 System, Promega, WI, USA), also employed in forensic genome identification, of which 18 loci were known to be heterozygous in OCI-AML3, fragment analysis of known NPM1^W288fs mutation [7 (link)] and qPCR of the DNMT3A^R882C mutation [8 (link)]. Median allele dropout was calculated by comparing replicate sample loci from STR. The described molecular analyses were performed in six single cell replicates and in triplicates for 2–50 cell input.

All cell lines used in this study were purchased from ATCC or identification was performed on established lines using PowerPlex® 21 System (Promega). Lines were tested for Mycoplasma every 3 months using the Venor®GeM Classic Mycoplasma PCR Detection Kit (Cambio Ltd). H1299, A549, Calu-6, H1703, H292, and Type II pneumocyte cells were maintained in RPMI 1640 medium, whereas Calu-1, HEK293, and iKRAS cells were grown in DMEM medium. An aliquot of 500 μM 4-OHT (48 h) and 100 ng/ml of doxycycline (48 h) were used to induce KRAS in Type II pneumocytes and iKRAS cells, respectively. MicroRNA mimics, anti-miRNAs, and siRNAs were purchased from Ambion. BID, RASA1, and NRAS Q61K plasmids were obtained from Addgene, KRAS wild type and KRAS^G12D were purchased from Origene.