DNA libraries of the three isolates, BS52, D2, and SI, were prepared with 1 µg of DNA using a TrueSeq DNA sample preparation kit (Illumina Cat. No. FC-121-2001). The DNA was fragmented using an ultrasonicator S220 (Covaris, Woburn, MA, USA) to obtain an average of 350 bp fragments. This was followed by end repair, A-tailing, ligation with Illumina adapters, size selection, and PCR amplification. The prepared libraries were quantified using Bioanalyzer 2100 (Agilent, Santa Clara, CA, USA) and quantitative PCR (qPCR). The clusters were generated using cBOT, and paired-end sequencing was performed using Illumina HiSeq 1000 platform (CCAMP, Bangalore, India). Sequencing data in the form of 2 × 100 bp paired-end reads were obtained. To improve the quality of the assembly, mate-pair sequencing of the D2 isolate was also performed using Illumina HiSeq 1000 platform (CCAMP, Bangalore, India).
Hiseq 1000 platform
Manufactured by Illumina
Sourced in India
The HiSeq 1000 platform is a high-throughput DNA sequencing system designed to produce large volumes of sequencing data. It utilizes Illumina's proprietary sequencing-by-synthesis technology to generate DNA sequence information. The HiSeq 1000 is capable of sequencing multiple samples simultaneously and producing gigabases of sequence data per run.
Automatically generated - may contain errors
Lab products found in correlation
2100 bioanalyzer, by Agilent Technologies (5 mentions)
Truseq rna sample preparation kit v2, by Illumina (4 mentions)
Truseq sbs kit v3 hs, by Illumina (3 mentions)
Qubit 2.0 fluorometer, by Thermo Fisher Scientific (2 mentions)
Bioanalyzer, by Agilent Technologies (2 mentions)
Rneasy kit, by Qiagen (2 mentions)
Turbo dna free dnase kit, by Thermo Fisher Scientific (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Nanodrop 1000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Spectrum plant total rna kit, by Merck Group (1 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
Agilent bioanalyzer, by Agilent Technologies (1 mentions)
Truseq exome enrichment kit, by Illumina (1 mentions)
Qiaamp dna blood midi kit, by Qiagen (1 mentions)
Truseq dna kit, by Illumina (1 mentions)
Tapestation 2200, by Agilent Technologies (1 mentions)
High sensitivity dna assay kit, by Agilent Technologies (1 mentions)
Nanodrop nd 8000 spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Mirneasy kit, by Qiagen (1 mentions)
29 protocols using hiseq 1000 platform
1
Preparation of Illumina DNA Libraries
2
Transcriptome Analysis of Nepenthes khasiana Leaf
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We extracted total RNA from all five distinct parts/zones of the N. khasiana leaf using Raflex Kit (Bangalore Genei, India) and/or Spectrum Plant Total RNA Kit (Sigma, USA) as per the instructions of the manufacturers. Quality check of the extracted RNA samples was performed using the Agilent 2100 Bioanalyzer. Extracted RNAs showed three distinct bands (28S, 18S and 5S) on EtBr-stained formaldehyde agarose gel (Additional file 1 : Fig. S15). RNA integrity number (RIN) values of the extracted RNAs ranged from 6.6–8.5. Library preparation and sequencing were performed at the Centre for Cellular and Molecular Platforms, Bangalore, India. One microgram of RNA each was used for library preparation and mRNA was purified using the polydT Oligo beads. The mRNA was then fragmented followed by cDNA synthesis. End repair, A-Tailing and adapter ligation were performed followed by PCR enrichment for 15 cycles. The Agilent Bioanalyzer was used to validate the generated libraries and sequencing was performed on an Illumina HiSeq 1000 platform as per the recommended protocol of the manufacturer, to generate 2 × 100 bp paired-end data. The five different parts/zones of the N. khasiana leaf were harvested from two separate individual plants and sequenced separately. Thus, two biological replicates were used for RNA-seq.
3
RNA-seq Analysis of Arabidopsis Transcriptome
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Triplicate cDNA libraries for each treatment were prepared and sequenced with 100 bp paired-end reads by the Illumina HiSeq 1000 platform at the Biotechnology Center in Akita Prefectural University. Raw data were preprocessed by three programs (FASTX-Toolkit, Prinseq and Trimmomatic). RNA-seq analysis was carried out using the Tophat-Cufflinks pipeline [28 (link)], with the following versions: Tophat v2.0.14, Bowtie2 v2.2.6.0 and Cufflinks v2.2.1. The Arabidopsis TAIR10 genome and gene model annotation data were downloaded and used for reference. Differentially expressed genes (DEGs) were determined by applying the screening thresholds of 20-fold changes in FPKM (fragments per kilobase of exon per million fragments mapped) and FDR-adjusted p-value ≤ 0.05 using Cuffdiff tool.
4
Whole Blood Exome Sequencing Protocol
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Genomic DNA was isolated from whole blood, using the QIAamp DNA Blood Midikit (Qiagen), according to the manufacturer’s protocol. Exonic regions of genomic DNA of three affected subjects (IDs III:5, III:8, and IV:4) were enriched using either the TruSeq™ Exome Enrichment Kit (Illumina) or the Agilent Haloplex Exome kit based on DNA digestion and capture. Exomes were barcoded and sequenced at multiple sites on the Illumina HiSeq1000 platform, and either 2 × 76-bp (TruSeq) or 2 × 100-bp (Haloplex) PE libraries, using TruSeq SBS Kit v3–HS (Illumina) reagents and a TruSeq PE Cluster kit v.3-cbot-HS flow cell. Average coverage for all the experiments was 70x and at least 20x for 89% of the target. Paired sequencing reads were aligned to the reference genome (UCSC, hg19 build) using BWA [16 (link)] and sorted with SAMtools [17 (link)] and Picard (http://broadinstitute.github.io/picard/ ). Post-alignment processing (local realignment around insertions-deletions and base recalibration), SNV, and small insertions-deletions (ins-del) calling were performed with Genome Analysis Toolkit (GATK) [18 (link)] with parameters adapted to the haloplex-generated sequences. The called SNV and ins-del variants produced with both platforms were annotated using ANNOVAR [19 (link)].
5
DNA Fragmentation and Sequencing Library Preparation
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DNA (500 ng) was fragmented through sonication using a Covaris S220 instrument (Covaris, Woburn, MA, USA) and DNA‐seq libraries were generated using the TruSeq DNA kit according to the manufacturer's instructions (Illumina, San Diego, CA, USA). Library length was assessed by capillary electrophoresis on a 2200 TapeStation (Agilent Technologies) and quantified by qPCR using primers annealing to the adapter sequences. DNA‐seq libraries were sequenced on an Illumina HiSeq1000 platform and generating 100‐bp paired‐end reads for a total of 2.5 Gb.
6
Comprehensive RNA Extraction and Sequencing
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Total RNA was extracted from the 50 human retina samples using the miRNeasy Kit (QIAGEN) according to the manufacturer's instructions. RNA was quantified using a NanoDrop ND-8000 spectrophotometer (NanoDrop Technologies) and the integrity was evaluated using an RNA 6000 Nano chip on a Bioanalyzer (Agilent Technologies). The RNA of the 50 samples had an average RNA integrity number (RIN) of 8.7 (ranging from 7.2 to 9.7). Libraries were prepared according to manufacturer's instructions (TruSeq RNA Sample Preparation kit) with an initial amount of 4 μg of total RNA. Quality control of library templates was performed using a High Sensitivity DNA Assay kit (Agilent Technologies) on a Bioanalyzer (Agilent Technologies). Qubit quantification platform was used to normalize samples for the library preparation (Qubit 2.0 Fluorometer, Life Technologies). Libraries were sequenced via a paired-end chemistry on an Illumina HiSeq1000 platform with an average yield of ∼6 Mb.
7
Genomic DNA Isolation and Sequencing
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Genomic DNA was isolated from Δhns-stpA and evolved strains using SIGMA GenElute™ Bacterial Genomic DNA Kit (Cat. No. NA2120) using the manufacturer's protocol. Sequencing library was prepared for Illumina sequencing and the quality of the libraries checked using Agilent Bioanalyzer at the genomics facility, Centre for Cellular and Molecular Platforms (C-CAMP), Bangalore. DNA isolated from HS100 and HS250 populations was sequenced for 50 cycles from one end, and that from the single colonies sequenced for 100 cycles from both ends. The sequencing was performed on the Illumina HiSeq1000 platform at C-CAMP. Raw data have been deposited with NCBI-SRA under the accession number SRP043310.
8
RNA-seq pipeline for transcriptome analysis
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Total RNA was isolated using Qiagen RNeasy Kit (catalog number 74106, Qiagen). RNA was quantified using a NanoDrop ND-8000 spectrophotometer (NanoDrop Technologies), and the integrity was evaluated using an RNA ScreenTape chip on an Agilent 2200 TapeStation. The RNA of the 16 samples had RNA integrity number above 9.5. Libraries were prepared according to manufacturer’s instructions (TruSeq RNA Sample Preparation kit) with an initial amount of 1 μg of total RNA. Quality control of library templates was performed using a High Sensitivity D1000 ScreenTape (catalog number 5067-5584, Agilent Technologies) on an Agilent 2200 TapeStation. Qubit quantification platform was used to normalize samples for the library preparation using Qubit double-stranded DNA HS Assay Kit (catalog number Q32854, Thermo Fisher Scientific–Life Technologies) on Qubit 2.0 Fluorometer, Life Technologies. Libraries were sequenced via a paired-end chemistry on an Illumina HiSeq 1000 platform. Sequencing reads from each one of the 16 samples were aligned to GENCODE Human transcripts on HG19 genome using bowotie2 aligner (35 (link)). The expression of each gene then was estimated in the form of expected counts using RSEM (36 (link)) and normalized for sequencing depth using the method developed in DESeq2 package (37 (link)) in the R statistical environment with default parameters.
9
Promoter Capture Hi-C in Mouse ESCs
10
Exome Sequencing of Tumor Samples
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DNA extracted from mononuclear cells and fresh tumor samples (containing at least 70% malignant cells) from eight patients was used to prepare a DNA library with the Illumina Nextera Rapid Capture Expanded kit (Illumina, Inc., San Diego, CA, USA/FC-140-1004), as detailed in Supplementary Methods. Shortly, genomic DNA (gDNA) was enzymatically fragmented while tags were simultaneously added. After purification, a limited-cycle PCR program was performed to ligate adapters and amplify libraries. Once gDNA libraries were prepared, exon-specific capture probes attached to streptavidin beads were used to enrich fragments containing only regions of interest, comprising 201,121 exons, totaling 62 mega base pairs (Mbp) of the genome. Exome libraries were then evaluated on a DNA 1000 Agilent 2100 Bioanalyzer chip (Agilent Technologies, Santa Clara, CA, USA) and quantified using KAPA SYBR FAST qPCR Kits (Kapa Biosystems, Wilmington, MA, USA, part #KK4602) prior to cluster generation. Pooled libraries were loaded on six lanes of one flow cell and sequenced on HiSeq 1000 platform (Illumina, Inc.) using 2 × 100bp paired-end reads, with a median of 95.3% of targeted bases covered at least 30-fold across the sample set.
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