RNA was extracted using the AllPrep Kit (Qiagen). Sequencing libraries for RNA-sequencing were prepared using TruSeq RNA Library Prep Kit V2 (Illumina). Paired-end 100 bp read sequencing was performed on a HiSeq 2500 system using Illumina TruSeq V3 chemistry. Paired-end reads were mapped to the human genome (NCBI build 37) by the gapped aligner STAR 2.4.117, using the two-pass method and parameters recommended by the NCI Genomic Data Commons (GDC) 18. The alignment file was used to calculate the raw digital gene expression values by HTseqcount software 0.7.219, using the intersection-nonempty model, which were further analyzed to provide digital gene expression values. The alignment file was also used for variant calling by VarScan2 with selection based on variant read count ≥ 3 and variant read frequency ≥ 0.1.
Truseq v3 chemistry
Manufactured by Illumina
Sourced in United States
The TruSeq v3 chemistry is a core sequencing reagent kit developed by Illumina. It is designed to enable high-quality DNA sequencing on Illumina's sequencing platforms. The kit includes the necessary reagents and enzymes required for sample preparation and sequencing reactions.
Automatically generated - may contain errors
Lab products found in correlation
Hiseq 2000, by Illumina (4 mentions)
Allprep kit, by Qiagen (2 mentions)
Truseq rna sample prep kit v2, by Illumina (2 mentions)
Hiseq 2000 platform, by Illumina (2 mentions)
Truseq rna library prep kit v2, by Illumina (1 mentions)
Total rna purification kit, by Norgen Biotek (1 mentions)
Hiseq 2000 system, by Illumina (1 mentions)
Smarter ultra low rna kit for sequencing, by Illumina (1 mentions)
Truseq pe cluster kit v3, by Illumina (1 mentions)
Nebnext ultra directional rna library prep kit, by Illumina (1 mentions)
2100 bioanalyzer, by Agilent Technologies (1 mentions)
Casava 1, by Illumina (1 mentions)
Rta 1, by Illumina (1 mentions)
Hiseq 2500 system, by Illumina (1 mentions)
11 protocols using truseq v3 chemistry
1
Comprehensive RNA-Seq and Variant Analysis
2
Exome Sequencing of Index Patient
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The index patient was exome sequenced by Oxford Gene Technology’s Genefficiency Sequencing Service. Genomic DNA (2 μg) was fragmented and enriched for human exonic sequences using the Human All Exon V5 Agilent Sure Select kit (Agilent Technologies) using the manufacturers protocol, and sequenced on the Illumina HiSeq 2000 platform using Truseq (v3 Chemistry) (Illumina) to generate 100 base paired-end reads. Fastq files were mapped to the reference human genome (hg19/GRCh37) using the Burrows-Wheeler Aligner (BWA) package (v0.6.2) [6 ]. Local realignment of the mapped reads around potential insertion/deletion (indel) sites was carried with the Genome Analysis Tool Kit (GATK) v1.6 [7 (link)]. Duplicate reads were marked using Picard v1.8 and BAM files were sorted and indexed with SAM tools v0.1.18 [8 ]. Approximately 12 and 14 GB of sequence data was generated for these samples and a minimum of 90.83% and 80.54% of the targeted exome was covered to a depth of at least 20× and 30× coverage, respectively. We filtered the variants for high-quality heterozygous, novel variants [defined against dbSNP 132 inclusion] and are deleterious based on either of the SIFT, Polyphen and Condel predictions.
Data cannot be shared publicly. The data underlying the results presented in the study are available from the corresponding author or from Dr. Per Hoffmann (Email:phoffmann@lifeandbrain.com ).
Data cannot be shared publicly. The data underlying the results presented in the study are available from the corresponding author or from Dr. Per Hoffmann (Email:
3
RNA-seq Analysis of Human Genome
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The total RNAs were isolated and used for high-throughput sequencing with TruSeq v3 chemistry (San Diego, CA 92121, USA) and 50 bp single reads on an Illumina HiSeq 2000. For RNA-seq analysis, sequenced reads were aligned to the Homo sapiens genome (version GRCh38.p8 from NCBI) using TopHat 2.1.1 [27 (link)] linked to Bowtie 2.2.8 [28 (link)] with the default sensitive settings. From the sequenced reads, transcripts were assembled using Cufflinks 2.2.1 [29 (link)]. Differential expression analyses were performed with Cuffdiff 2.2.1 [30 (link)] using a minimum false-discovery rate (qvalue) of <0.002 as statistical significance and log2 fold change >1.5 as the cut-off. Additional information for each gene was obtained from the NCBI database and included in the dataset. Genes with expression levels under a threshold in both the control and treatment conditions were discarded. The median of the distribution of the non-zero values was taken as the threshold. The generated data were uploaded to the FIESTA viewer FIESTA@BioinfoGP (https://bioinfogp.cnb.csic.es/tools/FIESTA/index.php )
4
Transcriptome Profiling of Drosophila-Herpetomonas Interaction
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Total RNA of 8–10 flies at 6h, 12h, 18h post H. muscarum oral infection was extracted with total RNA purification kit from Norgen Biotek following the manufacturer’s instruction. Each time point was repeated in three independent experiments. cDNA libraries were prepared with the Illumina TruSeq RNA Sample Prep Kit v2. All sequencing was performed on the Illumina HiSeq 2000 plaftform using TruSeq v3 chemistry (Oxford Gene Technology, OGT). All sequence was paired end and performed over 100 cycles. Read files (Fastq) were generated and then mapped to the concatenated D. melanogaster and H. muscarum genome sequences using the hisat2 mapper [98 (link)]. Mapped reads were then counted using HTseq-count (v. 0.10.0) [112 (link)] and differential expression analysed using the DESeq2 package in R [113 (link)].
5
High-Throughput Total RNA-Seq Analysis
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Total RNA-seq (~20 million reads, 75 bp paired end sequencing) was carried out by Oxford Gene Technology (Oxford, UK). Briefly, samples were prepared using the Illumina TruSeq RNA Sample Prep Kit v2 (Illumina, San Diego, CA, USA). Sequencing was performed on the Illumina HiSeq2000 platform using TruSeq v3 chemistry (Illumina, San Diego, CA, USA).
6
Poly(A) RNA-seq Library Preparation
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The polyadenylated fraction of RNA isolated from eight samples was used for 100bp paired-end RNA-seq with coverage 43.8 ± 4.1 sd million pairs read per sample. We used the SMARTer® Ultra™ Low RNA Kit for Illumina® Sequencing (Clontech) followed by the NEBNext® DNA Library Prep Master Mix Set for Illumina® to contruct poly(A) selected pair-end sequencing libraries. Both kits were used as per the manufacturer’s instructions except that published in-house custom indexes were used (45 (link)). The resulting multiplexed libraries were sequenced using Illumina TruSeq v3 chemistry. After indexing, all samples were combined into a single library and sequenced on two lanes of an Illumina HiSeq 2000 System.
7
RNA-seq Library Preparation and Analysis
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Poly-A selected libraries were prepared from total RNA at the Oxford Genomics Centre using NEBNext ultra directional RNA library prep kit for Illumina with custom 8bp indexes [70 (link)]. Libraries were multiplexed (3 samples per lane), clustered using TruSeq PE Cluster Kit v3, and paired-end sequenced (100nt) using Illumina TruSeq v3 chemistry on the Illumina HiSeq2000 platform. Samples were mapped with TopHat2 [71 (link)] on default settings with GENCODE v18 [19 (link)] as transcriptome and GRCh37 as genome reference. Exon level reads counts for all protein-coding and long non-coding transcripts present in GENCODE v18 were quantified with RNA-SeQC [72 (link)] with the “strictMode” flag set. Transcript level counts were compiled by adding up the counts for all exons. The sequenced data was required to contain at least 10M mapped and properly paired reads after applying the quality filters.
8
Total RNA Extraction and RNA-Seq Profiling
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Total RNA was extracted from each of the five samples using RNeasy Kit QIAGEN for animal cells and tissues (Qiagen, Nederland), including a DNase treatment. RNA molecules longer than 200 nucleotides were bound to a silica column membrane and eluted in RNase-free water. The quantity of RNA was measured with NanoDrop® ND-1000 UV–vis Spectrophotometer with an absorbance from about 200 nm up to 350 nm. Roughly 20 μg of total RNA were obtained for each sample. The quality of the RNA samples was tested with the Agilent Bioanalyzer 2100. The intensity ratio 28S/18S after the separation of total RNA on denaturing agarose gel electrophresis was around two which corresponds to good quality samples. RNA Integrity Numbers (RIN) for the different samples ranged between 5.8 and 7.4. The cDNA libraries -Truseq, v3 chemistry, with poly-A selection, were performed according to the manufacturer’s instructions (Illumina, San Diego, CA, USA), then sequencing was done on the Hiseq2000. The five cDNA libraries were sequenced in pair-ends, with reads of 101 bp, by the GATC company (Germany). Samples were sequenced in two different runs (GA and RA on one run, while the egg sample was sequenced in another run). The raw sequence data has been deposited in the SRA division of Genbank (project accession: PRJNA294954).
9
Illumina Library Prep with Inline Barcodes
10
Genomic Profiling of Single SKBR3 Cells
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A DNA fragment library was constructed from WGA products from individual SKBR3 cells picked from whole blood spike-in samples using a modified version of the NEBNext (New England Biolabs) protocol. Libraries were enriched using the SeqCap EZ Exome v3 capture system (Roche NimbleGen) for the coding portion of the genome. The target includes all coding content from the CCDS, RefSeq and miRBase databases. Paired-end (100 base pair) sequencing of enriched libraries was performed using a HiSeq 2500 system with TruSeq v3 chemistry (Illumina) with a read depth of 15 – 30x. The resulting reads were aligned to the genome human reference (hg19) using BWA (Burrows-Wheeler Aligner) [13 (link)] and variants called with GATK (Genome Analysis Toolkit) [14 (link),15 (link)].
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