The sequencing library was constructed using the QuantiSeq 3′ mRNA-Seq Library Prep Kit (Lexogen Inc., Vienna, Austria) according to the manufacturer’s instructions. Total RNA (500 ng) was extracted, and an Illumina platform-compatible sequence containing an oligo-dT primer was hybridized for reverse transcription. An Illumina-compatible linker sequence containing random sequences was used as the posterior procedure for degradation of the RNA template. Magnetic beads were used to purify the double-stranded sequencing library. Complete adapter sequences for cluster generation were added to amplify the library. The high-throughput sequencing procedure (as the single-end 75 sequences) was then performed using the NextSeq 500 device (Illumina Inc., San Diego, CA, United States). mRNA-seq analysis identified 23,282 genes. Changes in their expression levels were calculated as log2 values for genes with higher than normalized read count of four.
Nextseq 500 device
Manufactured by Illumina
Sourced in United States
The NextSeq 500 is a high-throughput sequencing system designed for a wide range of applications, including gene expression analysis, targeted resequencing, and small RNA sequencing. The device utilizes reversible terminator-based sequencing chemistry to generate high-quality sequencing data.
Automatically generated - may contain errors
Lab products found in correlation
Ribo zero, by Illumina (5 mentions)
Agilent sureselectxt reagent kit, by Agilent Technologies (4 mentions)
Sureselect all exon v5 or v6, by Agilent Technologies (4 mentions)
Nuclisens easymag kit, by bioMérieux (3 mentions)
Dragen covidseq test, by Illumina (3 mentions)
Covidseq test, by Illumina (3 mentions)
Maxwell 16 lev rna ffpe purification kit, by Promega (3 mentions)
Nextseq 500 550 high output kit v2.5 75 cycles, by Illumina (2 mentions)
Truseq stranded mrna kit, by Illumina (2 mentions)
Truseq stranded total rna library prep kit, by Illumina (2 mentions)
Nextseq 500 550 high output kit v2, by Illumina (2 mentions)
Emag device, by bioMérieux (2 mentions)
Truseq nano dna kit, by Illumina (1 mentions)
Cell free dna bct tube, by Streck (1 mentions)
Influx cell sorter, by BD (1 mentions)
Anti cd19 microbeads, by Miltenyi Biotec (1 mentions)
Single cell 5 library gel bead kit, by 10x Genomics (1 mentions)
Truseq dna pcr free library prep kit, by Illumina (1 mentions)
Hiseq 2500 device, by Illumina (1 mentions)
Qubit dna hs assay kit, by Thermo Fisher Scientific (1 mentions)
41 protocols using nextseq 500 device
1
Transcriptome Analysis Using QuantiSeq 3' mRNA-Seq
2
SARS-CoV-2 Genome Sequencing and Phylogenetic Analysis
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The full-length SARS-CoV-2 genomes from all included patients were sequenced by means of next-generation sequencing. Briefly, viral RNA was extracted from nasopharyngeal swabs in viral transport medium using NucliSENS® easyMAG kit on EMAG device (bioMérieux, Marcy-l’Étoile, France). Sequencing was performed with the Illumina COVIDSeq Test (Illumina, San Diego, CA, USA), which uses 98-target multiplex amplifications along the full SARS-CoV-2 genome. The libraries were sequenced with NextSeq 500/550 High-Output Kit v2.5 (75 Cycles) on a NextSeq 500 device (Illumina). The sequences were demultiplexed and assembled as full-length genomes by means of the DRAGEN COVIDSeq Test Pipeline on a local DRAGEN server (Illumina). Lineages and clades were interpreted using Pangolin and NextClade, before being submitted to the GISAID database (https://www.gisaid.org ; accessed on 1 June 2021; the GISAID IDs of the sequenced sampled are listed in the Supplementary Materials ). Phylogeny was performed after full-length genome alignment with Muscle v3.8.31 (maximum-likelihood model GTR + I; 1000 bootstrap replicates), by means of IQ-Tree v1.3.11.1 and iTOL.
3
Cell-free DNA Isolation from Maternal Plasma
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Approximately 10 ml of maternal blood in a Streck Cell-Free DNA BCT® tube was centrifuged at 1,600 × g for 10 min and then for another 10 min at 3,000 × g to isolate the plasma. Cell-free DNA was extracted from 1 ml of the isolated plasma using the Tiangen micro DNA kit (Tiangen Biotech Co., Ltd., Beijing, China), and the library was constructed using the TruSeq nano DNA kit (Illumina, San Diego, CA, United States). The NextSeq 500 device (Illumina) was used for sequencing at the 75-bp single-end mode, and approximately 12 million reads were generated per sample.
4
Panel-based Tumor DNA Sequencing
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Panel-based next generation sequencing was conducted in cases with DNA that passed quality controls. Tumor DNA was sequenced using a home-made panel of genes as previously described [23 (link)]. For each tumor sample, a library of all coding exons and intron–exon boundaries of a panel of 794 target genes (Additional file 1 : Table S1) was constructed using the SureSelect enrichment system (Agilent Technologies, Santa Clara, CA, USA). Sequencing was carried out using the Illumina NextSeq500 device (San Diego, CA, USA), according to the manufacturer’s instruction at a median depth of 162×.
5
Single-cell RNA-seq of Memory B Cells
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Single-cell suspensions from BM of 3× NP-CGG immunized female C57BL/6 mice were prepared and CD19+ cells were enriched by magnetic cell sorting using anti-CD19 microbeads (Miltenyi Biotech). Ex vivo IgG1+/IgG2b+CD19+CD38+GL7−CD138−IgM−IgD− memory B cells were isolated by FACS (Influx cell sorter (BD Bioscience)) and applied to the 10× Genomics platform using the Single Cell 5′ Library & Gel Bead Kit (10× Genomics) following the manufacturer’s instructions. The amplified cDNA was used for simultaneous 5′ gene expression (GEX) and murine BCR library preparation. BCR transcripts were amplified by Chromium Single Cell V(D)J Enrichment Kit for murine B cells (10× Genomics). Upon adapter ligation and index PCR, the quality of the obtained cDNA library was assessed by Qubit quantification, Bioanalyzer fragment analysis (HS DNA Kit, Agilent) and KAPA library quantification qPCR (Roche). The sequencing was performed on a NextSeq500 device (Illumina) using either a High Output v2 Kit (150 cycles) with the recommended sequencing conditions (read1: 26nt, read2: 98nt, index1: 8 nt, index2: n.a.) or a Mid Output v2 Kit (300 cycles for BCR repertoire analysis (read1: 150 nt, read2: 150 nt, index1: 8 nt, index2: n.a., 20% PhiX spike-in).
6
Whole-Genome Sequencing of Recombinant Strains
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Genomic DNA was extracted from selected putative recombinants, as well as from each donor and recipient strain, as previously described (83 (link)), and the resulting DNA samples were used for library preparation using the TruSeq DNA PCR-free library prep kit (Illumina) or the Nextflex PCR-free DNA-Seq kit for Illumina (Bioo Scientific). DNA sequencing was performed at the Biomics platform of the Institut Pasteur (Paris, France) using a paired-end 100-bp run on a HiSeq 2500 device (Illumina) or a paired-end 150-bp run on a NextSeq 500 device (Illumina). Sequencing reads were first trimmed using Trimmomatic v0.36 (84 (link)) (parameters LEADING 28, TRAILING 28, SLIDINGWINDOW windowSize 5, requiredQuality 28, MINLEN 50, and AVGQUAL 28). Complete read pairs were then de novo assembled using SPAdes v3.10.1 (85 (link)) (parameters --careful, -k 21,33,55 for HiSeq reads or -k 21,33,55,77 for NextSeq reads, and --phred-offset 33). The contigs thus generated were finally organized using MeDuSa v1.6 (86 (link)) (default parameters) and compared to the corresponding reference genome of each donor or recipient strain (see Table S4 in the supplemental material for details) (25 (link), 28 (link), 32 (link), 45 (link), 47 (link), 87 (link)– (link)89 (link)).
7
FFPE RNA-seq Transcriptome Analysis
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Total RNA was extracted from formalin-fixed paraffin-embedded (FFPE) tumor slices (5 × 5 µm) using the Maxwell 16 LEV RNA FFPE Purification kit (Promega) following manufacturer instructions. Libraries were prepared from 12 µl of total RNA with the TruSeq Stranded Total RNA using Ribo-Zero (Illumina) following manufacturer instructions. Once qualified, paired-end libraries were sequenced using 2 × 75 bp output on a NextSeq 500 device (Illumina).
The abundance of transcripts from RNA-seq data was quantified through the Kallisto program.24 (link) This program is based on pseudo alignment for rapidly determining the compatibility of reads with targets, without the need for alignment. The Kallisto transcript index used as reference was built from merged human cDNA and ncDNA files from GRCh37 assembly ENSEMBL. Gene-level count matrices were then created with the DESeq2 library. Low-count genes were pre-filtered by removing genes with too few reads.25 (link) Enrichr software was used to analyze Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways.26 (link),27 (link)
The abundance of transcripts from RNA-seq data was quantified through the Kallisto program.24 (link) This program is based on pseudo alignment for rapidly determining the compatibility of reads with targets, without the need for alignment. The Kallisto transcript index used as reference was built from merged human cDNA and ncDNA files from GRCh37 assembly ENSEMBL. Gene-level count matrices were then created with the DESeq2 library. Low-count genes were pre-filtered by removing genes with too few reads.25 (link) Enrichr software was used to analyze Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways.26 (link),27 (link)
8
Single-Cell Genomics and Immune Profiling
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Single cell suspensions were obtained by cell sorting and applied to the 10x Genomics workflow for cell capturing and scRNA gene expression (GEX) and TCR/BCR/CiteSeq library preparation using the Chromium Single Cell 5′ Library & Gel Bead Kit as well as the Single Cell 5′ Feature Barcode Library Kit (10x Genomics). After cDNA amplification the CiteSeq libraries were prepared separately using the Single Index Kit N Set A. TCR/BCR target enrichment was performed using the Chromium Single Cell V(D)J Enrichment Kit for Human T cells and B cells, respectively. Final GEX and TCR/BCR libraries were obtained after fragmentation, adapter ligation, and final Index PCR using the Single Index Kit T Set A. Qubit HS DNA assay kit (Life Technologies) was used for library quantification and fragment sizes were determined using the Fragment Analyzer with the HS NGS Fragment Kit (1–6000 bp) (Agilent).
Sequencing was performed on a NextSeq500 device (Illumina) using High Output v2 Kits (150 cycles) with the recommended sequencing conditions for 5′ GEX libraries (read1: 26nt, read2: 98nt, index1: 8nt, index2: n.a.) and Mid Output v2 Kits (300 cycles) for TCR/BCR libraries (read1: 150nt, read2: 150nt, index1: 8nt, index2: n.a., 20% PhiX spike-in).
Sequencing was performed on a NextSeq500 device (Illumina) using High Output v2 Kits (150 cycles) with the recommended sequencing conditions for 5′ GEX libraries (read1: 26nt, read2: 98nt, index1: 8nt, index2: n.a.) and Mid Output v2 Kits (300 cycles) for TCR/BCR libraries (read1: 150nt, read2: 150nt, index1: 8nt, index2: n.a., 20% PhiX spike-in).
9
RNA-seq analysis of tumor-infiltrating CD8+ T cells
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Tumor-infiltrating CD8 T cells from CT26-tumor bearing mice treated with Glucose 5% (control) or Folfox when tumors reached 50–70 mm2 were sorted by flow cytometry 8 days following treatment. CD8 TILs cells coming from 2 independent experiments with 8–10 pooled tumors for each experiment were used. Splenic CD8 T cells from 2 naïve mice were used as reference. Total mRNA was isolated using Trizol (Gibco Life Technologies) according to the manufacturer's instructions. rRNA from total RNA extracted were removed with NEBNextrRNA depletion kit (New England BioLabs). 100 ng of RNA depleted of rRNA was used for the library preparation with a NEBNext Ultra RNA library kit for Illumina according to the manufacturer's instructions (New England BioLabs). RNA sequencing was performed on a NextSeq 500 device (Illumina). The libraries were sequenced with paired-end 75–base pair ‘reads’. FASTQ files were mapped with the BWA software package (mm10 National Center for Biotechnology Information assembly of the Mus musculus genome) for Illumina. Analysis was performed with the splice junction mapper TopHat for Illumina. The files generated were processed with Cufflinks software to obtain annotated expressed genes in each studied subtype. Heatmaps of selected genes were generated using R software (http://www.R-project.org ).
10
RNA-seq Transcriptome Analysis of Cell Samples
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Following MACS, cells were immediately lysed and total RNA was extracted using the RNeasy® Micro Kit (Qiagen). Library preparation and Illumina sequencing were performed at the Ecole normale supérieure GenomiqueENS core facility (Paris, France). Messenger (polyA+) RNAs obtained in three independent experiments (biological replicates) were purified from 150 ng of total RNA using oligo(dT). All samples had RNA integrity numbers ranging from 9,1 to 9,9 as assessed by the Fragment Analyzer instrument (Agilent Technologies). Libraries were prepared using the strand-specific RNA-Seq library preparation TruSeq Stranded mRNA kit (Illumina). Libraries were multiplexed by twelve on a run. A 75 bp single-end read sequencing was performed on NextSeq 500 device (Illumina). A mean of 33 ± 1.7 million reads passing the Illumina quality filter was obtained for each of the 12 samples.
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