Two separate M. marinum infection experiments were prepared for RNA-seq. Total RNA was isolated by the Ambion PureLink™ RNA Mini kit in combination with TRIzol (Invitrogen), followed by on column DNase treatment according to the manufacturer’s instructions (Ambion, Invitrogen). RNA yield and quality were measured by Nanodrop (Thermo Scientific) and by agarose gel electrophoresis, respectively. Preparation of L. pneumophila infected samples were described previously [23 (link)]. Total RNA samples, biological duplicates for each time point, were sent to SciLifeLab Stockholm for library preparation and high-throughput sequencing. mRNA was isolated from the total RNA pool using poly(A) extraction with oligo dT prior to sequencing with Illumina HiSeq 2000/2500 system.
Hiseq 2000 2500 system
Manufactured by Illumina
Sourced in China, Canada, United States
The HiSeq 2000/2500 system is a high-throughput DNA sequencing platform developed by Illumina. It is designed to perform massively parallel sequencing of DNA samples, enabling rapid and efficient generation of large amounts of sequence data.
Automatically generated - may contain errors
Lab products found in correlation
2100 bioanalyzer, by Agilent Technologies (2 mentions)
Truseq sbs kit v3 hs 200 cycles reagents, by Illumina (2 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Purelink rna mini kit, by Thermo Fisher Scientific (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Nanodrop nd 1000 spectrophotometer, by Agilent Technologies (1 mentions)
Novaseq 6000 system, by Illumina (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Truseq small rna sample prep kit, by Illumina (1 mentions)
Ribo zero rrna removal kit, by Illumina (1 mentions)
Sureselect human all exon v4 kit, by Agilent Technologies (1 mentions)
Bigdye terminator v1, by Thermo Fisher Scientific (1 mentions)
Haloplex target enrichment kit, by Agilent Technologies (1 mentions)
Truseq sbs kit v3, by Illumina (1 mentions)
Nebnext end repair da tailing module, by New England Biolabs (1 mentions)
Kapa real time amplification kit, by Roche (1 mentions)
Nebnext ultra ligation module, by New England Biolabs (1 mentions)
Nebnext ultra 2 dna library prep kit, by Illumina (1 mentions)
Humanht 12 v4.0 expression beadchip, by Illumina (1 mentions)
Isolate 2 rna mini kit, by Meridian Bioscience (1 mentions)
23 protocols using hiseq 2000 2500 system
1
RNA-seq Profiling of M. marinum Infection
2
Comprehensive RNA Expression Analysis
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The total RNA of the ‘leaf’ and ‘pet’ samples was isolated and purified using Trizol reagent (Invitrogen, CA, USA). The content and quality of the RNA were assessed using a NanoDrop ND-1000 spectrophotometer (Wilmington, DE, USA) and an Agilent 2100 Bioanalyzer (CA, USA).
To analyze the expression of mRNAs, lncRNAs, and circRNAs, a Ribo-Zero™ rRNA Removal Kit (Illumina, San Diego, USA) was used to remove ribosomal RNA for construction of the chain-specific library. Each of the ‘pet’ and ‘leaf’ samples comprised three biological replicates, and hence a total of six libraries (‘leaf 1,’ ‘leaf 2,’ ‘leaf 3,’ ‘pet 1,’ ‘pet 2,’ and ‘pet 3’) were prepared. The qualified libraries were sequenced using the Illumina NovaSeq™ 6000 system (LC-BIO, China). The read length of the double-ended sequence was 2 × 150 bp.
A total of six small RNA libraries was prepared using TruSeq Small RNA Sample Prep Kits (Illumina, San Diego, USA) to analyze miRNA expression. The prepared libraries were sequenced using the Illumina HiSeq 2000/2500 system (LC-BIO, China). The single sequence read length was 1 × 50 bp.
To analyze the expression of mRNAs, lncRNAs, and circRNAs, a Ribo-Zero™ rRNA Removal Kit (Illumina, San Diego, USA) was used to remove ribosomal RNA for construction of the chain-specific library. Each of the ‘pet’ and ‘leaf’ samples comprised three biological replicates, and hence a total of six libraries (‘leaf 1,’ ‘leaf 2,’ ‘leaf 3,’ ‘pet 1,’ ‘pet 2,’ and ‘pet 3’) were prepared. The qualified libraries were sequenced using the Illumina NovaSeq™ 6000 system (LC-BIO, China). The read length of the double-ended sequence was 2 × 150 bp.
A total of six small RNA libraries was prepared using TruSeq Small RNA Sample Prep Kits (Illumina, San Diego, USA) to analyze miRNA expression. The prepared libraries were sequenced using the Illumina HiSeq 2000/2500 system (LC-BIO, China). The single sequence read length was 1 × 50 bp.
3
ChIP-seq and RNA-seq Profiling of Histone Modifications
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A ChIP assay was completed as previously described [36 (link)], with antibodies specific for H3 trimethyl-Lys 27 (Upstate, USA), H3 trimethyl-Lys 4 (Abcam), H3 trimethyl-Lys 36 (Abcam), H3 dimethyl-Lys 9 (Abcam), H3 monomethyl-Lys 4 (Upstate), H3 acetyl-Lys 27 (Upstate), and H3 acetyl-Lys 9 (Upstate). For each ChIP-sequencing assay, approximately 30 seedlings were pooled together and ground to a powder. More than 10 ng ChIP DNA, 2 μg mRNA and 2 μg total RNA (rRNA depleted) were used to prepare each sequencing sample. Libraries were constructed and sequenced by Genenergy Biotechnology Co. Ltd. (Shanghai, China). For total RNA-seq, the strand-specific library was constructed as previously described [37 (link)]. The libraries were sequenced with the HiSeq 2000/2500 system (Illumina) to produce 150-bp paired-end reads. The sample preparation, library construction, and sequencing were completed with two biological replicates. Sequencing data for the replicates had relatively high Pearson correlation coefficients (Additional file 1 : Figure S6). The ChIP-sequencing data were highly correlated with the data from a recently published study [13 (link)], which involved a ChIP-sequencing analysis of H3K27me3, H3K4me3, and H3K9ac (Additional file 1 : Figure S7).
4
Illumina-based Genomic Variation Analysis
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The DNA sequences of each cultivar were analysed by an Illumina HiSeq 2000/2500 system (Illumina Co, Ltd.)38 (link),39 (link) and mapped to Os-Nipponbare-Reference-IRGSP-1.040 (link),41 (link). We identified a total of 670,069 single nucleotide polymorphisms (SNPs) and InDels after removing nucleotide variations with missing rates > 0.10 and a minor allele frequency < 0.025. To illustrate the population structure from genotype data, we performed principal component analysis using the R function “prcomp”.
5
Genetic Analysis of Ancient Tooth
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An ancient tooth was used in the genetic analysis. DNA extraction was performed using 120 mg of tooth powder. All procedures were carried out in room facilities dedicated to analysing ancient DNA. DNA was extracted from tooth powder along with blank control, and DNA extract was converted into single-stranded DNA libraries15 (link). The amplified and indexed libraries were sequenced on an Illumina HiSeq 2000/2500 system (for details, see SI).
6
Genetic Screening for Hereditary Spastic Paraplegia
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Genomic DNA was extracted from the peripheral blood according to standard procedures.9 (link) For a subset of the cohort from the province of Ontario (37 patients), all exons and flanking intron sequences of a panel of 51 genes known to cause HSP were sequenced both in forward and reverse directions at the Hospital for Sick Children using next-generation sequencing techniques. Sanger validation was performed according to standard protocols using BigDye terminator v1.1 (Life Technologies, Carlsbad, CA), and sequencing products were separated on an Applied Biosystems model 3730 Capillary Sequencer (Life Technologies) and analyzed using SeqPilot software (JSI Medical Systems, Kippenheim, Germany). Whole-exome sequencing was performed on 108 samples from 51 families without a genetic diagnosis, using the Agilent SureSelect Human All Exon v4 kit for capture and targeted enrichment of the exome. The captured samples were sequenced in the Illumina HiSeq 2000/2500 system (Genome Québec Innovation Centre, Montreal, QC, Canada). The reads were then aligned against the human genome (GRCh37 assembly) using Burrows-Wheeler Aligner.10 (link) Variant calling and annotation were performed using Genome Analysis ToolKit11 (link) and Annotate Variation.12 (link) The exome data were then screened for all known HSP-causing genes and mutations.13 (link),14 (link)
7
Cancer Gene Resequencing and Mutation Detection
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The exons of 872 genes related to cancer were selected for resequencing (Supplementary Table S1 ). Target capture was performed using 200 ng of DNA and reagents from a HaloPlex Target Enrichment kit (Agilent), according to the Automation Protocol Version D.3. The 172 samples in the diagnostic cohort and the 24 relapse samples were enriched individually. Remission samples were enriched in pools of ten samples. The 165 samples in the extension cohort were enriched in pools of ten samples without a unique individual barcode for each sample. However, each sample was present in two pools using a design that enabled assignment of rare somatic mutations to their carrier (Lindqvist et al, manuscript in preparation). In addition, 84 samples from healthy Swedish blood donors were enriched in pools of 21 samples and used for filtering purposes. Paired-end sequencing with 100 bp reads was performed on a HiSeq2000/2500 system (Illumina). The average sequence depth per sample in the target region was 638× for ALL samples enriched individually (diagnostic cohort and relapse samples), 529× for ALL samples in pools (extension cohort), 162× for remission samples and 133× for Swedish blood donors.
8
Illumina Paired-end Sequencing Protocol
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All libraries were sequenced by using the Illumina HiSeq 2000/2500 system with TruSeq SBS Kit v3-HS (200 cycles) reagents (part no. FC-401-3001) to generate paired-end 101–base pair reads.
9
Illumina Sequencing Library Preparation
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About 0.5–5 ng of cDNA or ChIP‐precipitated DNA were used as starting material for the generation of sequencing libraries with the NEBNext Ultra II DNA library prep kit for Illumina (NEB). Alternatively, sequencing libraries were generated using the NEBNext End Repair/dATailingModule and NEBNext Ultra Ligation Module (NEB), followed by amplification with the KAPA Real‐Time Amplification kit (KAPA Biosystems). Cluster generation and sequencing were carried out using the Illumina HiSeq 2000/2500 system according to the manufacturer's guidelines. Dataset EV3 provides further information about all sequencing experiments of this study.
10
Comprehensive Genetic Profiling of Dementia
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Genomic DNA was extracted from peripheral blood samples using a standard extraction method. The whole-exome sequencing (WES) and targeted panels comprising 168 genes potentially associated with AD and other types of dementia were used to perform comprehensive genomic testing. Sequencing was performed using the Illumina Hiseq2000/2500 system. Sequenced data analysis was carried out as described previously (14 (link)). Sanger sequencing was used to validate the candidate variants after variant calling and filtering. Co-segregation analysis of variants was performed in the families with available DNA samples.
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