Wild-type mice were co-transferred with Foxo1+/+ ERT2cre B1-8hi (CD45.1+CD45.2−) and Foxo1f/f ERT2cre B1-8hi (CD45.1+CD45.2+) B cells, and immunized with NP-CGG precipitated in alum on day 0. On day 7, 8, and 9, mice were orally administered tamoxifen. On day 12, Foxo1+/+ LZ (CD45.1+CD45.2−B220+NP+GL7+CD38−CD86hiCXCR4lo) and DZ GC B cells (CD45.1+CD45.2–B220+NP+GL7+CD38−CD86loCXCR4hi), and Foxo1f/f GC B cells (CD45.1+CD45.2−B220+NP+GL7+CD38−) were sorted for RNA preparation. Three biological replicates were used in each genotype. Construction of DNA libraries for RNA-seq and sequencing were performed as described previously (Shinnakasu et al., 2016 (link)). In brief, the DNA library was constructed using an NEBNext Ultra RNA Library Prep kit for Illumina (NEB) from total RNA purified from ∼104 sorted cells. RNA-sequencing was performed on a HiSeq 1500 sequencer (Illumina) in a 49-bp single-end read mode. Gene set enrichment analysis was performed using GSEA software v2.2.2 (Subramanian et al., 2005 (link)). The lists of gene signature of up-regulated genes by CD40 and BCR stimulation for gene set enrichment analysis were previously described (Victora et al., 2010 (link); Shinnakasu et al., 2016 (link)). The RNA-seq data are available at Gene Expression Omnibus database under accession no. GSE93554 .
Hiseq 1500 sequencer
Manufactured by Illumina
Sourced in United States
The HiSeq 1500 is a high-throughput DNA sequencing system designed for a variety of genomic applications. It utilizes sequencing by synthesis technology to generate high-quality sequence data. The HiSeq 1500 is capable of producing up to 1 terabase of sequence data per run, making it a powerful tool for large-scale genomic projects.
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Lab products found in correlation
Ampure xp bead, by Beckman Coulter (2 mentions)
Agencourt rnaclean xp beads, by Beckman Coulter (2 mentions)
Kapa hifi hotstart readymix, by Roche (2 mentions)
Rneasy kit, by Qiagen (2 mentions)
Nanodrop 1000, by Thermo Fisher Scientific (2 mentions)
Nebnext ultra rna library prep kit for, by Illumina (1 mentions)
Dynabeads mrna purification kit, by Thermo Fisher Scientific (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
2100 bioanalyzer, by Agilent Technologies (1 mentions)
Nextera xt dna sample preparation kit, by Illumina (1 mentions)
Truseq dna kit, by Illumina (1 mentions)
Qubit system, by Thermo Fisher Scientific (1 mentions)
Tapestation 2200, by Agilent Technologies (1 mentions)
D1000 screentape, by Agilent Technologies (1 mentions)
Qubit dsdna hs assay kit, by Thermo Fisher Scientific (1 mentions)
Accel ngs 1s plus dna library kit for, by Illumina (1 mentions)
Turbo dnase, by Thermo Fisher Scientific (1 mentions)
4 thiouridine 4su, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
27 protocols using hiseq 1500 sequencer
1
Foxo1 Regulation in Germinal Center B Cells
2
Genome Sequencing and Assembly Protocol
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The sequencing libraries with insert sizes of 170, 250, and 500 base pairs (bp) were prepared for sequencing of the paired-end reads, following a modified version of the manufacturer's protocol (Illumina, San Diego, CA, USA). An integrated protocol from the Mate-Pair Library v2 Sample Preparation Guide (Illumina) and the Paired-End Library Preparation Method Manual (Roche, Branford, Connecticut, USA)) was used to prepare mate-pair libraries with insert sizes of 3, 6, 10, 15, and 20 kb (Additional file 1: Table S1b, S1c ). For the transcriptome sequencing, Poly(A)+ messenger RNA (mRNA) was purified using the DynaBeads mRNA Purification kit (Life Technologies, Carlsbad, CA). Paired-end complementary DNA libraries were constructed using the RNA sequencing (RNA-Seq) Next-Generation Sequencing Library Preparation Kit for Whole-Transcriptome Discovery (Gnomegen, San Diego, CA). All of the libraries are sequenced on an Illumina HiSeq 1500 sequencer. The D. mawsoni genome was assembled using SOAPdenovo (SOAPdenovo, RRID:SCR_010752 ) [17 (link)] to build the contigs and SSPACE (SSPACE, RRID:SCR_005056 ) [18 (link)] to scaffold the contigs. The E. maclovinus genome was assembled using Platanus (Platanus, RRID:SCR_015531 ) to build the contigs, and SSPACE to scaffold the contigs.
3
RNA-seq Analysis of Rat Transcriptome
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Library construction and RNA sequencing were performed by the Marshall University Genomics Core Facility. RNA libraries were prepared for each sample using the Truseq RNA mRNA library prep kit from Illumina. RNA sequencing (2 × 50 paired end reads; 20 million reads/sample) was performed on an Illumina HiSeq1500 sequencer.
RNA sequencing reads were aligned to the rat genome rn06 obtained from Ensembl, using HISAT2 version 2.1.0 (Kim et al., 2015 (link)), and the resulting BAM files were sorted using SAMtools version 1.9 (Li et al., 2009 (link)). Read quality was checked using FastQC (Andrews, 2010 ). Reads were trimmed to remove low‐confidence base calls and adapter sequences using Trimmomatic version 0.38 (Bolger et al., 2014 (link)), and then mapped to known transcripts from Ensembl genes version 94 using the R/Bioconductor package Genomic Alignments version 1.16.0 (Lawrence et al., 2013 (link)). Differentially expressed genes were identified using DESeq2 version 1.20.020 with false discovery rate (Benjamini–Hochberg adjusted p‐value) <0.1 used as a threshold for statistical significance (Benjamini & Hochberg, 1995 ).
RNA sequencing reads were aligned to the rat genome rn06 obtained from Ensembl, using HISAT2 version 2.1.0 (Kim et al., 2015 (link)), and the resulting BAM files were sorted using SAMtools version 1.9 (Li et al., 2009 (link)). Read quality was checked using FastQC (Andrews, 2010 ). Reads were trimmed to remove low‐confidence base calls and adapter sequences using Trimmomatic version 0.38 (Bolger et al., 2014 (link)), and then mapped to known transcripts from Ensembl genes version 94 using the R/Bioconductor package Genomic Alignments version 1.16.0 (Lawrence et al., 2013 (link)). Differentially expressed genes were identified using DESeq2 version 1.20.020 with false discovery rate (Benjamini–Hochberg adjusted p‐value) <0.1 used as a threshold for statistical significance (Benjamini & Hochberg, 1995 ).
4
Transcriptomic Analysis of Galectin-3 Knockdown
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HeLa cells were transfected with siRNA to silence galectin-3 and with the non-silencing control siRNA against firefly luciferase. Total RNA was isolated with the RNeasy Mini Kit (Qiagen) and processed by the TruSeq® Stranded mRNA LT Kit to prepare RNA-seq libraries. The libraries were sequenced on an Illumina HiSeq 1500 sequencer via paired-end sequencing to obtain 2×50 bp paired reads. The obtained reads were mapped against the Homo sapiens genome reference (Ensemble Revision 74, hg19) using the STAR algorithm [23 (link)]. FPKM values were calculated for each sample, differential gene expression was analyzed using DEseq2 [24 (link)]. The genes with a FPKM value above 0.3 in at least one sample and a DEseq p-value of 0.05 or better were considered as differentially expressed if the absolute of the log2 fold change was one or larger. Differential exon usage was analyzed using DEXseq [25 (link)]. All algorithms used standard parametrization.
5
Illumina Library Preparation for Viral Sequencing
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An Illumina library was generated using the Illumina Nextera XT DNA sample preparation kit. Briefly, it involved fragmentation of the DNA molecules, and addition of the adapters with a sequence barcode. The quality and size of the DNA library were visualized and quantified using the Agilent Bioanalyzer 2100 instrument. No size selection was carried out. Sequencing was carried out using the Illumina HiSeq1500 sequencer in high-output mode using a 2 × 101 sequencing run according to the manufacturer’s instructions. A total of six sequencing runs were carried out involving EBV, RSV, FeLV, and REO1 (three spiking levels and two different freeze-thaw conditions).
6
ChIP-seq Library Preparation Protocol
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ChIP samples were subjected for ChIP-seq library preparation using the TruSeq DNA kit (Illumina, FC-121- 2001) and Agencourt AMPure XP beads (Beckman coulter, A63881) with slight modifications. After end repair, A-tailing and adaptor ligation, DNA fragments were purified with two volumes of beads. Purified DNA was amplified by 18–22 cycle of PCR cycle and size selection was performed. Greater than 370 bp DNA fragments were removed with 0.7 volume of beads, these selected DNA was recovered with 1.8 volume of beads, and >250 bp DNA fragments were collected with 1.0 volume of beads.
DNA length was analysed on a D1000 screen tape (Agilent, 5067-5582) using D1000 sample buffer (Agilent, 5067-5583) on an Agilent 2200 tape station. DNA quantity was analysed on a Qubit dsDNA HS assay kit (Invitrogen, Q32851) using Qubit system (Invitrogen). Single-end 50 bp sequencing was performed for ChIP-seq libraries of egg extract-treated sperm and blastula DNA, and paired-end 32 bp sequencing for ICeChIP-seq libraries of Xenopus and human sperm DNA on a HiSeq 1500 sequencer (Illumina).
DNA length was analysed on a D1000 screen tape (Agilent, 5067-5582) using D1000 sample buffer (Agilent, 5067-5583) on an Agilent 2200 tape station. DNA quantity was analysed on a Qubit dsDNA HS assay kit (Invitrogen, Q32851) using Qubit system (Invitrogen). Single-end 50 bp sequencing was performed for ChIP-seq libraries of egg extract-treated sperm and blastula DNA, and paired-end 32 bp sequencing for ICeChIP-seq libraries of Xenopus and human sperm DNA on a HiSeq 1500 sequencer (Illumina).
7
ChIP-Seq Library Prep and Analysis
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ChIP sample library preparations from >4 ng of ChIP-DNA was performed using Accel-NGS 1S Plus DNA Library Kit for Illumina (Swift Biosciences). A 50 bp single-end sequencing was done with the Illumina HiSEQ 1500 sequencer to a sequencing depth of ~70 million reads. Fastq-files were mapped against the human genome (hg19, GRCh37, version 2009), extended for the EBV genome (NC007605) using bowtie (v1.1.1) (Langmead et al., 2009 (link)). Sequencing profiles were generated using deepTools’ bamCoverage function using reads extension to 200 bp and reads per genomic content normalization (Ramírez et al., 2016 (link)). Visualization was performed in UCSC Genome Browser (http://genome.ucsc.edu ).
For H4K20me1 and -me3 ChIP-seq data, MACS2 peak-calling (Zhang et al., 2008 (link)) was performed using the broad setting and overlapping peaks in three replicates were retained for further analyses.
For H4K20me1 and -me3 ChIP-seq data, MACS2 peak-calling (Zhang et al., 2008 (link)) was performed using the broad setting and overlapping peaks in three replicates were retained for further analyses.
8
Jurkat Cell Activation and Transcriptome Analysis
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Jurkat cells were acquired from DSMZ (Braunschweig, Germany). Cells were grown in RPMI 1640 medium (Gibco) supplemented with 10% heat‐inactivated FBS (Gibco) and 1% penicillin/streptomycin (100×, PAA) at 37°C under 5% CO2. Cells were labeled in media for 5 min with 500 μM 4‐thiouridine (4sU, Sigma‐Aldrich) and activated with 50 mM PMA (Sigma‐Aldrich) and 1 μM ionomycin (Sigma‐Aldrich). Cells were harvested, spike‐ins were added, and RNA was purified and fragmented as described (Schwalb et al, 2016 ). Fragmented RNA was subjected to purification of labeled RNA as described (Dölken et al, 2008 ). Labeled fragmented RNA (TT‐seq) and total fragmented RNA (Total RNA‐seq) were treated with 2 units of DNase Turbo (Life Technologies). Sequencing libraries were prepared with the Ovation Human Blood RNA‐seq library kit (NuGEN) following the manufacturer's instructions. All samples were sequenced on an Illumina HiSeq 1500 sequencer.
9
Single-cell RNA-seq library preparation
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Library preparation was done as described in22 (link) with the following modifications: 6N unique molecular identifiers were added to the 5' end of the 3' sequencing adapter; primers and adapters concentrations were lowered to match the low RNA input; Agencourt RNAClean XP beads (Beckman Coulter) were used for clean-up and size separation; pre-adenylated 5' and 3' adapters were used and all ligation reactions were carried without ATP to reduce ligation artefacts. All libraries and controls went through 13 PCR cycles using KAPA HiFi HotStart Ready Mix (KAPA Biosystems). PCR products were size-selected on a 1.8% agarose gel before loading on a HiSeq 1500 sequencer (Illumina).
10
Comprehensive SARS-CoV-2 Genome Sequencing
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Total RNA was extracted from bronchial samples using the DSP virus/pathogen Mini Kit on a Qiasymphony robot (Qiagen, Hilden, Germany). Amplicon pools spanning the whole virus genome were prepared using the ARTIC protocol multiplex PCR, subsequently converted to barcoded sequencing libraries with the Nextera XT kit (Illumina, San Diego, USA) and sequenced on an Illumina Hiseq1500 sequencer (Illumina, San Diego, USA) as described previously [27 (link)]. The sequenced amplicons were demultiplexed and consensus sequences were generated using the iVar pipeline. The pileup files served as input for the consensus sequence generation within iVar where only variants that had a minimum read depth of 20 and a minimum frequency of 0.9 were considered. Phylogenetic analyses were achieved with the web and analysis platform Auspice using the SARS-Cov-2 build (https://github.com/nextstrain/ncov ) and the bioinformatic toolkit augur. The consensus sequences and meta data for the samples were uploaded to the GISAID repository. GISAID accession numbers for the cases of this study are shown in Table 2 .
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