Formaldehyde-crosslinking of cells, sonication, DNA extraction of FAIRE-enriched fractions and Illumina library preparation was performed as previously described [19 (link)]. Libraries were prepared in triplicate from infected and mock-infected samples at 1, 12, 24 and 48 h, using the Illumina TruSeq Sample Prep kit, and were sequenced on the Illumina 2500 platform (101 bp paired-end read protocol) at the Genome Resource Centre, Institute for Genome Sciences, University of Maryland School of Medicine. Sequence data are available from the NCBI GEO archive GSE132448.
2500 platform
Manufactured by Illumina
Sourced in United States, China
The 2500 platform is a high-throughput sequencing system designed for a variety of applications. It offers a range of features and capabilities for DNA and RNA sequencing.
Automatically generated - may contain errors
Lab products found in correlation
Ecori, by New England Biolabs (3 mentions)
Nuclean plant genomic dna kit, by CWBIO (2 mentions)
2100 bioanalyzer, by Agilent Technologies (2 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (2 mentions)
Qiaquick gel extraction kit, by Qiagen (2 mentions)
Nebnext dna library prep kit, by New England Biolabs (2 mentions)
Nebnext multiplex oligos for kit, by Illumina (2 mentions)
Truseq sample prep kit, by Illumina (1 mentions)
Dna extraction kit, by Tiangen Biotech (1 mentions)
Trizol, by Tiangen Biotech (1 mentions)
Truseq chip sample preparation kit, by Illumina (1 mentions)
Nebnext ultra rna library prep kit, by Illumina (1 mentions)
Superscript 2 reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Rnase h, by Thermo Fisher Scientific (1 mentions)
Dna polymerase 1, by Thermo Fisher Scientific (1 mentions)
Hiseq 2000 sequencing system, by Illumina (1 mentions)
Rna fragmentation kit, by Thermo Fisher Scientific (1 mentions)
Minielute column, by Qiagen (1 mentions)
Mycoalert plus mycoplasma detection kit, by Lonza (1 mentions)
Kapa hifi hotstart dna polymerase, by Roche (1 mentions)
27 protocols using 2500 platform
1
FAIRE-seq Sequencing of Infected Cells
2
Genomic DNA Extraction and Sequencing of A. giraldii Pamp.
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We collected fresh A. giraldii Pamp. Leaves from the Institute of Medicinal Plant Development (IMPLAD), Beijing, China. Then, the total genomic DNA (accession number: implad201910017) was extracted using a DNA extraction Kit (Tiangen Biotech, Beijing, China) and stored in a refrigerator at − 80 °C. A DNA sequence library was constructed with 1 ug of DNA by using a NEBNext library building kit and sequenced with a 2500 platform (Illumina, San Diego, CA, USA). Clean data were obtained by removing low-quality sequences with Trimmomatic software46 (link) under the following conditions: sequences with more than 50% bases with quality values (Q) of < 19 and more than 5% ‘N’ bases. The plant sample used for Illumina short‐read sequencing was subsequently used for Oxford Nanopore sequencing. Raw reads obtained by Nanopore sequencing were filtered to remove reads with Q of < 7. Genomic DNA was prepared using the CTAB method and purified with a QIAGEN genomic kit (Cat# 13343, QIAGEN) according to the standard operating procedure provided by the manufacturer. About 700 ng of DNA was used in library construction and then sequenced on a Nanopore PromethION sequencer instrument (Oxford Nanopore Technologies, UK) at the Genome Center of Grandomics (Wuhan, China).
3
Transcriptome Profiling of Leg Muscle
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Total RNA was extracted from each leg muscle sample with Trizol (TianGen Biotech, Beijing, China). The qualified RNA was used to construct the cDNA library, which was sequenced on an Illumina 2500 platform after quality inspection.
4
ChIP DNA Sequencing Library Preparation
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MAR DNA is already in the range of 50–100 bp, and very similar to ChIP DNA in size. Sequencing libraries were prepared using Illumina TruSeq ChIP sample preparation kit according to manufacturer’s instructions. Libraries were sequenced by single-end sequencing with 75 bp read length on the Illumina 2500 platform.
5
Chilling-Induced Differential Gene Expression
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Sequencing libraries were constructed using the NEBNext UltraTM RNA Library Prep Kit for Illumina according to the manufacturer’s instructions. PCR was performed with size-selected, adaptor-ligated cDNA using Phusion High-Fidelity DNA polymerase, and the products were purified (AMPure XP system). The sequencing was performed on an Illumina 2500 platform, and paired-end reads were generated.
The clean reads were mapped to rice reference genome (Rice Genome Annotation Project Release 7) using Hisat2 tools [54 (link)]. The significant differentially expressed genes (DEGs) were determined using DESeq2 [55 (link)]. |log2(FC)| ≥ 1 and FDR ≤ 0.01 were used as thresholds to identify chilling-induced DEGs.
The mRNA sequencing data of this study are available in Genome Sequence Archive in National Genomics Data Center, China National Center for Bioinformation/Beijing Institute of Genomics, Chinese Academy of Sciences (GSA: CRA005306).
The clean reads were mapped to rice reference genome (Rice Genome Annotation Project Release 7) using Hisat2 tools [54 (link)]. The significant differentially expressed genes (DEGs) were determined using DESeq2 [55 (link)]. |log2(FC)| ≥ 1 and FDR ≤ 0.01 were used as thresholds to identify chilling-induced DEGs.
The mRNA sequencing data of this study are available in Genome Sequence Archive in National Genomics Data Center, China National Center for Bioinformation/Beijing Institute of Genomics, Chinese Academy of Sciences (GSA: CRA005306).
6
RNA-seq Analysis of Intestinal Epithelial Cells
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RNA-seq libraries were prepared from IECs and sequenced using the Illumina 2500 platform. The sequencing procedure followed the manufacturer’s protocols, generating raw sequencing data in FASTQ format for further analysis. The raw sequencing data underwent preprocessing to remove adapter sequences, low-quality reads, and potential contaminants. Trimmomatic (v0.39) was utilized for this purpose. Subsequently, the processed reads were aligned to the mouse reference genome (mm10) using STAR (v2.5.3a), and gene expression quantification was performed with HTSeq (0.11.0). The resulting read counts were normalized using the DESeq2 package. Differential expression analysis was conducted using edgeR. To gain insights into the biological implications of the differentially expressed genes, gene ontology enrichment analyses were performed utilizing the R package clusterProfiler (v4.0.5).
7
Urothelial Cell Transcriptome Profiling
8
Whole Exome Sequencing for Variant Detection
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Whole exome sequencing (WES) was undertaken using an Illumina 2500 platform. Libraries were prepared by SureSelect kit (Illumina Inc., San Diego, California). Captured sequences were read on the system and aligned using in‐house pipeline. Generated data were analyzed using publicly and commercially available tools and software. After the analysis of the WES data, the variants were filtered as previously described.9 , 10 , 12 , 13 , 14
9
Illumina-based RNA-Seq Protocol for Transcriptomics
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Sample DL3 was used for RNA-Seq analysis. A sequencing library for the Illumina 2500 platform was created from the polyadenylated fraction of RNA. mRNA was isolated with Dyna1 oligo-dT beads (Thermo Fisher Scientific, Waltham, MA) from 10 μg of total RNA. The mRNA was randomly fragmented using the RNA Fragmentation kit from Ambion. First-strand cDNA synthesis was performed using random primers and SuperScriptII Reverse-Transcriptase (Thermo Fisher Scientific, Waltham, MA). This was followed by second-strand cDNA synthesis using DNA Polymerase I and RNase H (Thermo Fisher Scientific, Waltham, MA). The Illumina adaptor was ligated to the ends of the double-stranded cDNA fragments and a 200 bp size-selection of the final product was performed by gel-excision, following the Illumina-recommended protocol. 200 bp adapter-ligated cDNA template fragments were enriched by PCR to create the final library. The DL3 RNA-Seq library was sequenced at the New York Genome Center in one lane on the Illumina HiSeq 2000 Sequencing System, using 2 × 100bp reads.
10
mRNA Extraction and RNA-seq Library Preparation
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The mRNA extraction, cDNA library construction and sequencing were performed by the Beijing Center for Physical and Chemical Analysis (BCPCA) (Beijing, China). Total RNA was extracted from each sample using the TRlzol reagent and digested with DNase I. Oligo (dT) magnetic beads were used to enrich mRNA from the total RNA and then broken into short fragments by a fragmentation buffer. To build the cDNA libraries based on the four samples (Solexa/Illumina 2,500 platform: 101 bp short insert-paired end), we followed the methods of Neeraja et al. (2012 (link)) and Tang et al. (2011 (link)).
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