Hiseq 2500 or 4000

Detailed descriptions of patient samples/methods are presented in supplementary methods, available at Annals of Oncology online. Briefly, peripheral blood samples were collected from 254 patients with ER+ BC, plasma was isolated from 20 ml whole blood, ≥20 ng DNA was extracted, and hybrid capture-based genomic profiling of ctDNA was carried out in a CLIA-certified/CAP-accredited laboratory [Foundation Medicine (FM)] to identify substitutions, short insertions/deletions, rearrangements/fusions, and amplifications [12 ]. Sixty-two genes (supplementary Table S1, available at Annals of Oncology online) were sequenced (Illumina HiSeq 2500 or 4000) to a median unique coverage depth of 7503×. Maximum somatic allele frequency (MSAF) was used to estimate the ctDNA fraction in plasma.

NPs were dissociated using 0.05% Trypsin (Gibco) spun down in ES-medium, resuspended, washed, and spun down in 10 ml PBS (Gibco). Afterwards, cells were resuspended in 1 ml N2B27 and filtered into a FACS tube (Falcon). The Fluidigm C1 platform was used to capture individual cells using 96 small or medium IFC chips. Cells were diluted in the range of 250,000–400,000 cells per ml for chip loading. Capturing efficiency was evaluated by manually inspecting each capture site on the chip using the automated NanoEntek JuLi cell imager. Only capture sites containing single cells were processed for library preparation and sequencing. Single cell full-length cDNA was generated using the Clontech SMARTer Ultra Low RNA kit on the C1 chip using manufacturer-provided protocol. ArrayControl RNA Spikes (AM1780) were added to the cell lysis mix, as recommended in the Fluidigm protocol. Libraries were prepared using the Illumina Nextera XT DNA Sample Preparation kit, according to a protocol supplied by Fluidigm, and sequenced on Illumina Hiseq 2500 or 4000 using 50- or 75-bp paired-end runs.

DNAs from each sorted tumor population and a patient-matched control sample were sequenced within the Mayo Clinic Medical Genome Facility (MGF) by using established protocols for whole exome analysis. Briefly, whole exon capture was carried out with Agilent’s SureSelect Human All Exon 71 MB version 6 kit; 500 ng of the prepped library is incubated with whole exon biotinylated RNA capture baits supplied in the kit for 24 h at 65 °C. The captured DNA:RNA hybrids are recovered by using Dynabeads MyOne Streptavidin T1 (Thermo Fisher Scientific, Waltham, MA, USA). The DNA was eluted from the beads and desalted by using purified Ampure XP beads (Beckman Coulter Life Sciences, Indianapolis, IN, USA). The purified capture products were amplified by using the SureSelect Post-Capture Indexing forward and Index polymerase chain reaction (PCR) reverse primers (Agilent Technologies) for 12 cycles. Libraries were loaded onto paired-end flow cells at concentrations of 4–5 pM to generate cluster densities of 600,000–800,000/mm² by using the Illumina cBot and HiSeq Paired-end cluster kit version 3 (Illumina, San Diego, CA, USA). The flow cells are sequenced as 101×2 paired-end reads on an Illumina HiSeq 2500 or 4000 by using TruSeq SBS sequencing kit version 3 and HiSeq data collection version 1.4.8 software. Base-calling was performed by using Illumina’s RTA version 1.12.4.2.