Maxwell system

The sample origins were as follows: (1) for WM tumor cells: bone marrow at diagnosis in all patients; (2) for DLBCL cells: spleen (patient 1), inguinal lymph node (patient 2) and bone marrow (patients 3 and 4) at transformation; and (3) for germline cells: peripheral blood samples in which no infiltration (<0.1%) was demonstrated by flow cytometry.
DNA was extracted by conventional methods: manually with the DNAzol reagent (MRC, Cincinnati, OH, USA) or automatically with the Maxwell system (Promega Corporation, Madison, WI, USA). Quantification and quality control of DNA were evaluated before enrichment and library preparation. DNA concentrations were measured using the Qubit fluorometer (Thermo Fisher Scientific, Inc., Waltham, MA, USA). DNA sample quality was assessed by gel electrophoresis. At least 1 μg of DNA was required for library preparations.

Male mice with deletion of the gene Sry from the Y-chromosome, but expressing Sry transgene on an autosome (termed FCG mice) aged 8–12 weeks were bred to females of 3 different backgrounds (Ldlr^−/−, Apoe^−/−, and C57BL/6 J) to generate male and female mice with an XY or an XX sex chromosome complement. Mice genotypes were identified by amplifying DNA extracted from tail or ear clips using a Promega Maxwell system and polymerase chain reaction (PCR) using a commercial PCR mix (Promega 2X Master Mix, cat#m7123) and specific primers for the Sry transgene, presence of the Y-chromosome, and internal positive control. Ldlr^−/− FCG mice (12–16 weeks old) were fed Western diet, 42% kcal from fat (TD88137, Harlan Teklad, Indianapolis, IN), Apoe^−/− FCG mice (14–16 weeks old) were fed standard murine diet (Purina 5001; approximately 5% fat, PMI Nutrition International, St. Louis, MO), and C57BL/6 J FCG mice (14–16 weeks old) were fed an atherogenic diet (7.5% cocoa butter, 1.25% cholesterol, 0.5% sodium cholate, TD90221, Harlan Teklad, Indianapolis, IN) for 16 weeks. All experiments were approved by the animal care and use committee at the University of Kentucky and the University of California, Los Angeles and conformed to the Guide for the Care and Use of Laboratory Animals published by the NIH.

DNA was extracted from FFPE material. Using H&E staining, areas with the highest tumor cell content were selected and DNA was extracted automatically, using a Maxwell system (Promega, Fitchburg, WI, USA) and the Maxwell 16 FFPE Plus LEV DNA Purification Kit according to the manufacturer’s instructions. The DNA concentrations were determined with the Invitrogen Qubit dsDNA BR Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA), and a FLUOstar Omega Microplate Reader (BMG Labtech GmbH, Ortenberg, Germany).