Cell free dna screentape assay

Mammary gland from 8-week-old mice (one p53^R172H/R172H knock-in mouse and one p53^+/+ mouse) were isolated and digested as previously described^{98 (link)}. scRNAseq libraries were prepared by using Chromium Next GEM Single Cell 3’ Reagent Kits v3.1 Dual Index (10X Genomics) following manufacturer’s instructions. Estimated 16,500 cells were loaded to each channel with the average recovery rate of 10,000 cells. Briefly, cells were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM). The poly-A RNA from the cell lysate contained in every single GEM was retrotranscribed to cDNA, which contains an Illumina R1 primer sequence, Unique Molecular Identifier (UMI) and the 10x Barcode. The pooled barcoded cDNA was then cleaned up with Silane DynaBeads, amplified by PCR and the appropriate size fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction Illumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added. The final indexed libraries were quantified by using the Cell-free DNA Screen Tape Assay (Agilent) on a TapeStation 4150 (Agilent) and were sequenced on HiSeqX (Illumina).

Pretreatment blood was collected in Cell-Free DNA BCT^®CE tubes and shipped immediately to Osaka University Hospital at ambient temperature. Tubes were centrifuged at 2000× g for 10 min and the supernatant was collected. Then, the supernatant was centrifuged again at 16,000× g for 10 min and the supernatant was collected and preserved at −80 °C. CfDNA was extracted from this supernatant with a MagMAX™Cell-Free DNA Isolation Kit according to the manufacturer’s instructions. The extracted DNA was quantified using a Qubit 1x dsDNA HS Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). The quality and quantity of cfDNA was assessed with the Cell-Free DNA Screen Tape Assay (Agilent Technologies, Santa Clara, CA, USA).

Peripheral blood samples were collected into EDTA vacutainer tubes at the time of hospital admission before infusion of IVIG and biologics. Plasma samples were collected by centrifuging at 1,600g for 10 minutes at 4°C, aliquoted to 1 mL volume in Eppendorf tubes, and stored at –80°C until use. Plasma was thawed and then centrifuged at high speed (16,000g) for 10 minutes at 4°C to remove residual debris. Plasma was spiked with fragmented unmethylated lambda DNA (725 copies/mL) to calculate extraction efficiency and the bisulfite conversion rate. Then, plasma cfDNA was isolated on an automated nucleic acid sample preparation QIAsymphony^SP (QIAGEN) instrument using the QIAsymphony DSP Circulating DNA kit according to the manufacturer’s protocol. The extracted cfDNA was eluted in 60 μL in LoTE buffer, quality checked using the Cell-free DNA ScreenTape assay on the 4150 TapeStation System (Agilent Technologies), and stored at –20°C for further use.