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Nextseq 500 550 high output kits v2

Manufactured by Illumina
Sourced in United States

The NextSeq 500/550 High Output Kits v2 are laboratory equipment designed for high-throughput DNA sequencing. The kits provide the necessary reagents and consumables to perform sequencing runs on the NextSeq 500 or NextSeq 550 platforms. The core function of these kits is to generate high-quality sequencing data by enabling the amplification, detection, and analysis of DNA samples.

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11 protocols using nextseq 500 550 high output kits v2

1

CD4+ T Cell Isolation and RNA-Seq

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CD4+ T cells were isolated from LNs of CD4-Cre (n=4) and Pou2af1fl/fl x CD4-Cre (n=9) mice after immunization with SRBCs by magnetic separation using CD4+ T cell isolation kit (Milteny Biotec). Purity of isolated CD4+ T cells was ≥ 96% for all samples and is shown in Figure S14. Extraction and purification of RNA were performed using RNeasy Mini Kit (Qiagen). RNA was eluted in RNase-free water and stored at -80°C. RNA quantity was measured using the Infinite 200 Pro (Tecan).
Quality was checked using an Agilent Tape Station and all samples displayed RIN values above 9. After quantity measurement by Qubit Fluorometer (Invitrogen) 200ng of RNA were used to generate sequencing libraries using the Illumina RNA-Seq Kit V2. Sequencing libraries were subsequently quality checked on D1000 screentapes (Agilent) and 10 samples each were sequenced using a NextSeq550 (Illumina) and 2 NextSeq 500/550 High Output Kits v2.5 (75 Cycles) at the Genomics Core Facility (Ulm University).
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2

Single-cell RNA-seq of Mesenchymal Cells

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Single-cell library preparation was performed using Chromium Single Cell 3′ GEM Library & Gel Bead Kit v3 (cat. no. 1000092, 10x Genomics) according to manufacturer’s instruction. The libraries were sequenced on the NextSeq 500 system (Illumina) using the NextSeq 500/550 High Output kits v.2.5 (50 cycles; cat. no. FC-404-2005, Illumina). The sequencing outputs were processed using the CellRanger software v.3.1.0 on the GHPCC. Reads were mapped to human reference genome GRCh38 (Ensembl 93). Data analysis was performed using Seurat v.4.1.0 (ref. 60 (link)) within R v.4.0.2 environment. RNA velocity analysis was performed using the velocyto v.0.17 commend line tool and velocyto.R v.0.6 R package61 (link). Sequencing results have been deposited in the Gene Expression Omnibus (accession no. GSE198482) and analysis scripts are available at https://github.com/zingery/mesenchymal-maintenance/.
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3

Targeted Gene Sequencing from Blood and Buccal Samples

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Each participant provided either 2 mL of peripheral blood or a buccal swab sample. Genomic DNA was extracted from blood samples by the GeneJet whole blood genomic DNA purification minikit (ThermoFisher, USA), or from buccal swab samples by the QIAamp DNA minikit (Qiagen, Germany). DNA fragmentation and library preparation were performed using the NEBNext Ultra II FS DNA library prep kit (New England Biolabs, USA) following the manufacturer’s instructions. Libraries were pooled together and hybridized with predesigned probes for 17 targeted genes (Integrated DNA Technologies, USA). Massive parallel sequencing was performed using NextSeq 500/550 High output kits v2 (150 cycles) on the Illumina NextSeq 550 system (Illumina, USA) with the minimum target coverage of 100x.
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4

Targeted NGS Sequencing of HBA Genes

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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.
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5

Targeted cfDNA and FFPE Sequencing

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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.
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6

scRNAseq Analysis of Tumor Immunity

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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.
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7

Single-Cell RNA-Seq of Lung Tumor-Infiltrating Leukocytes

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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.
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8

scRNA-seq of B[a]P-Induced Lung Tumors

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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.
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9

Single-Cell RNA-Seq of B(a)P-Induced Lung Tumors

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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.
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10

Tumor-infiltrating Leukocyte Profiling

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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 37oC 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.
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