Nextseq 500 550 high output kits v2

NGS library was prepared from cfDNA using NEBNext^® Ultra™ II FS DNA Library Prep Kit for Illumina (New England BioLabs, Ipswich, MA, USA) according to the manufacturer’s instructions. DNA library concentrations were quantified with a QuantiFluor^®dsDNA system (Promega, USA). Equal amounts of libraries (150 ng per sample) were pooled and hybridized with xGen Lockdown probes targeting coding exons of HBA1 and HBA2 (IDT DNA, USA). Sequencing was performed using NextSeq 500/550 High output kits v2 (2 × 75 cycles) on the NextSeq 550 system (Illumina, USA) with minimum target coverage of 12X. The mean coverage in the target regions for all samples was approximately 1852X.

For cell free DNA (cfDNA), library with unique molecular identifier tagging were prepared from 2 ng of cfDNA using the Accel-NGS 2S Plus DNA library kit (Swift Biosciences, USA) following the manufacturer’s instructions. Library concentrations were quantified with a QuantiFluor dsDNA system (Promega, USA). Equal amounts of libraries were pooled together and hybridized with xGen Lockdown probes for four targeted genes EGFR, KRAS, NRAS, BRAF (IDT DNA, USA). Sequencing was performed using NextSeq 500/550 High output kits v2 (150 cycles) on Illumina NextSeq 550 system (Illumina, USA) with the coverage of 10.000X.
For genomic DNA from FFPE, libraries were prepared from 2 ng of cfDNA using the NEBNext Ultra II FS DNA library prep kit (New England Biolabs, USA) following the manufacturer’s instructions. Similar to ctDNA libraries, FFPE libraries were pooled before hybridization with the xGen Lockdown probes and sequencing in the Illumina NextSeq 550 system. Both cfDNA and FFPE samples exhibited 5–10% on-target reads.

For scRNAseq, randomly selected lymph node samples (1 sample from CpG control group and 2 samples from TOP2A vaccine treated group) and the breast tumor samples (1 sample from CpG control group and 1 sample from TOP2A vaccine treated group) of C3(1)/Tag mice were harvested at the end of the study, minced and digested at 37 °C for 30 minutes with mouse tumor dissociation buffer (Miltenyi Biotec, CA) to generate single-cell suspensions per the manufacturer’s instructions. The processed samples were directly stained with violet viability dye, APC anti-CD45, and CD45+ leukocytes were sorted using FACS. FACS-sorted CD45+ leukocytes were then spun down at 300 g for 5 minutes and counted manually with a Neubauer Chamber. Approximately 2.0 × 10⁴ cells were loaded onto the 10X Chromium Controller per the manufacturer’s instruction, resulting in a recovery of about 1 × 10⁴ cells. For the lymph node samples, the libraries of single-cell transcriptome were generated by Chromium Single Cell 3’ v3 Reagent Kits (10x Genomics). For tumor samples, single-cell transcriptome and single-cell TCR libraries were prepared using a 10x Chromium Single-cell 5′ and VDJ library construction kit. All of the libraries were sequenced using NextSeq 500/550 High Output Kits v2 (150 cycles) (Illumina) according to the manufacturer’s protocol.

For scRNA‐seq, right middle lung lobes were harvested from mice in each treatment group at the end of the study, then minced and digested at 37 °C for 30 min with mouse tumor dissociation buffer (Miltenyi Biotec, CA) to generate single‐cell suspensions per the manufacturer's instructions. TILs were directly stained with violet viability dye, APC anti‐CD45, and CD45⁺ leukocytes were FACS sorted out. FACS‐sorted CD45⁺ leukocytes were then spun down at 300 × g for 5 min, counted manually with Neubauer Chamber. Approximately 2.0 × 10⁴ cells were loaded onto the 10x Chromium Controller per the manufacturer's instruction, resulting in a recovery of about 1 × 10⁴ cells. The scRNA‐seq libraries were generated by Chromium Single Cell 3' v3 Reagent Kits (10x Genomics) and sequenced using NextSeq 500/550 High Output Kits v2 (150 cycles) (Illumina) according to the manufacturer's protocol. There were two replicates for each experimental group.

For scRNA‐seq, B[a]P‐induced primary lung tumors were harvested and pooled from each mouse at the end of the study, then minced into 1–2 mm³ pieces, and digested at 37 °C for 20 min with mouse tumor dissociation buffer (Miltenyi Biotec, CA) to generate single‐cell suspensions. Red blood cells were lysed with ammonium–chloride–potassium (ACK) buffer, single‐cell suspensions were stained with 7‐AAD and CD45 on ice for 30 min, and CD45− and CD45+ populations were sorted by flow cytometry. For single‐cell library preparation, flow‐sorted CD45− or CD45+ cells were pelleted by centrifugation at 300 × g for 5 min and counted manually using a Neubauer Chamber. Approximately 1.6 × 10⁴ cells were loaded onto the 10X Chromium controller as per the manufacturer's instructions. The scRNA‐seq libraries were generated by Chromium single cell 3′ v3 reagent Kits (10x Genomics) and sequenced using NextSeq 500/550 high output kits v2 (150 cycles) (Illumina) according to the manufacturers’ protocols.

For scRNA-seq, B((a)P-induced primary lung tumors were harvested and pooled from each mouse at the end of the study, then minced into 1-2 mm³ pieces and digested at 37°C for 20 min with mouse tumor dissociation buffer (Miltenyi Biotec, CA) to generate single-cell suspensions. Red blood cells were lysed with Ammonium-Chloride-Potassium (ACK) buffer, single-cell suspensions were stained with 7-AAD and CD45 on ice for 30 min, and CD45- and CD45+ populations were sorted by flow cytometry. For single-cell library preparation, flow-sorted CD45- or CD45+ cells were pelleted by centrifugation at 300 g for 5 min and counted manually using a Neubauer Chamber. Approximately 1.6 × 10⁴ cells were loaded onto the 10x Chromium controller per the manufacturer’s instructions. The scRNA-seq libraries were generated by Chromium single cell 3’ v3 reagent Kits (10x Genomics) and sequenced using NextSeq 500/550 high output kits v2 (150 cycles) (Illumina) according to the manufacturer’s protocols.

For scRNA-seq, VC-induced primary lung tumors were harvested and pooled from different treatment groups at the end of the study, then minced and digested at 37^oC for 20 min with mouse tumor dissociation buffer (MiltenyiBiotec, CA) to generate single cell suspensions per the manufacturer’s instructions. Tumor-infiltrating leukocytes were directly stained with 7-AAD, CD45, and CD3 surface markers, and CD3⁺ or CD45⁺ TILs were flow-sorted. Flow sorted TILs were spin down at 300 × g for 5 min, counted manually with Neubauer Chamber. About 1.6 × 10⁴ cells were loaded onto the 10X Chromium Controller per the manufacturer’s instructions, resulting in recovery of about 1 × 10⁴ cells. The scRNA-seq libraries were generated by Chromium Single Cell 3′ v3 Reagent Kits (10× Genomics) and sequenced using NextSeq 500/550 High Output Kits v2 (150 cycles) (Illumina) according to the manufacturer’s protocol. There were two replicates for each experimental group.