Illumina-based mRNA-seq libraries were prepared from 1 μg RNA following previously published methods3 (link). mRNA was purified from total RNA using Sera-mag oligo(dT) magnetic beads (GE Healthcare Life Sciences) and then fragmented in the presence of divalent cations (Mg2+) at 94 °C for 6 min. The resulting fragmented mRNA was used for first-strand cDNA synthesis using random hexamers and reverse transcriptase, followed by second-strand cDNA synthesis using DNA polymerase I and RNaseH. Double-stranded cDNA was end-repaired using T4 DNA polymerase, T4 polynucleotide kinase and Klenow polymerase. The DNA fragments were then adenylated using Klenow exo-polymerase to allow the ligation of Illumina Truseq HT adapters (D501–D508 and D701–D712). All enzymes were purchased from Enzymatics. Following library preparation, quality control and quantification were performed using a 2100 Bioanalyzer instrument (Agilent) and the Quant-iT PicoGreen dsDNA Reagent (Invitrogen), respectively. Libraries were sequenced using Illumina HiSeq4000 sequencers to generate 50-bp single-end reads.
Truseq ht adapters
Manufactured by Illumina
Sourced in United States
The TruSeq HT adapters are a set of oligonucleotide sequences designed for use with Illumina's sequencing platforms. The adapters enable the attachment of DNA fragments to the flow cell surface, allowing for subsequent sequencing. The adapters are offered in a high-throughput (HT) format, providing increased sample multiplexing capabilities.
Automatically generated - may contain errors
Lab products found in correlation
Kapa hyper prep kit, by Roche (3 mentions)
Hiseq 4000 sequencer, by Illumina (2 mentions)
Sera mag oligo dt magnetic beads, by GE Healthcare (2 mentions)
Quant it picogreen dsdna reagent, by Thermo Fisher Scientific (2 mentions)
2100 bioanalyzer instrument, by Agilent Technologies (2 mentions)
Dneasy blood and tissue kit, by Qiagen (2 mentions)
Miseq platform, by Illumina (2 mentions)
Hiseq 2500, by Illumina (1 mentions)
Difco pseudomonas isolation agar, by BD (1 mentions)
Nebnext ultra 2 kit, by New England Biolabs (1 mentions)
Nucleospin gdna clean up kit, by Macherey-Nagel (1 mentions)
7 protocols using truseq ht adapters
1
Illumina-based mRNA-seq Library Preparation
2
RNA-seq Analysis of Medicago Plant Samples
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RNA from 138 samples was sequenced by the University of Minnesota's Genomic Center using Illumina HiSeq2500 100‐bp single‐end reads. One sample required resequencing (L88), which resulted in 125‐bp reads. Samples were barcoded and multiplexed using Illumina TruSeq HT adapters. Fastq files were checked with Fastqc version 0.11.5, and adapters were trimmed using cutadapt version 1.8.1 with non‐default parameters ‐m 40 and ‐q 30 (Andrews, 2010 ; Martin, 2011 ). Reads were then aligned to 4.0 gene models, and reference (http://jcvi.org/medicago/ ) using STAR 2.5.3a (Dobin et al., 2013 ), then filtered based on unique mapping scores, sorted and indexed using samtools version 1.6 (Li et al., 2009 ). FPKM values were generated using Cufflinks version 2.2.1 using non‐default parameters of ‐I 20000 and ‐‐min‐intron length 5. Raw sequencing files are publicly available on the NCBI SRA (PRJNA327225 and PRJNA449544).
3
Bacterial Genome Sequencing and Assembly
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Bacterial colonies were isolated on Difco Pseudomonas Isolation Agar (BD, Sparks, MD). Genomic DNA was extracted from overnight cultures using the DNeasy Blood and Tissue Kit (QIAGEN, Hilden, Germany). Genomic DNA (500 ng) was mechanically fragmented for 40 s using a Covaris M220 (Covaris, Woburn, MA) with default settings. Fragmented DNA was transferred to a polymerase chain reaction tube and library synthesis was performed with the Kapa Hyperprep kit (Kapa Biosystems, Wilmington, MA) according to the manufacturer’s instructions. TruSeq HT adapters (Illumina, SanDiego, CA) were used to barcode the libraries, which were each sequenced in 1/48 of an Illumina MiSeq 300-bp paired-end run at the Plateforme d’Analyses Génomiques of the Institut de Biologie Intégrative et des Systèmes (Université Laval, Québec, Canada). Each data set was assembled de novo with the A5 pipeline version A5-miseq 20140521. Raw reads, assembly data and metadata were uploaded on IPCD (https://ipcd.ibis.ulaval.ca ).
4
Genome Sequencing of Strains RP1 and RP45
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The genome of RP73 was previously published [19 (link)]. Genomic DNA from strains RP1 and RP45 was isolated from overnight cultures using the DNeasy Blood and Tissue Kit (QIAGEN). Genomic DNA (500 ng) was mechanically fragmented for 40 s using a Covaris M220 (Covaris, Woburn MA, USA) with default settings. Fragmented DNA was transferred to PCR tubes and library synthesis was performed with the Kapa Hyperprep kit (Kapa biosystems, Wilmington MA, USA) according to manufacturer’s instructions. TruSeq HT adapters (Illumina, SanDiego, CA, USA) were used to barcode the samples and each library was sequenced in 1/48 of an Illumina MiSeq 300 bp paired-end run at the Plateforme d’Analyses Génomiques of the Institut de Biologie Intégrative et des Systèmes (Laval University, Quebec, Canada). Sequencing data for each genome was assembled with the A5 pipeline [51 (link)]. Whole genome shotgun projects has been deposited at DDBJ/EMBL/GenBank under accessions LNBU00000000 (RP1) and LNDM00000000 (RP45).
5
Library Preparation for Short-Read Sequencing
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The library preparation protocol for short reads sequencing was as follows. Genomic DNA (500 ng in 55 ul TE buffer) was mechanically fragmented for 40 s using a Covaris M220 (Covaris, Woburn MA, USA) with default settings. Fragmented DNA was transferred to PCR tubes and library synthesis was performed using a NEB Next Ultra II kit (New England Biolabs) according to the manufacturer’s instructions. To barcode the samples, TruSeq HT adapters (Illumina, SanDiego, CA, USA) were used. The library was sequenced on the Illumina MiSeq platform (300-bp paired-end reads). Of the 15 335 342 raw paired-end reads obtained, 11 121 576 remained after elimination of low-quality reads.
6
Illumina-based mRNA-seq Library Preparation
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Illumina-based mRNA-seq libraries were prepared from 1 μg RNA following previously published methods3 (link). mRNA was purified from total RNA using Sera-mag oligo(dT) magnetic beads (GE Healthcare Life Sciences) and then fragmented in the presence of divalent cations (Mg2+) at 94 °C for 6 min. The resulting fragmented mRNA was used for first-strand cDNA synthesis using random hexamers and reverse transcriptase, followed by second-strand cDNA synthesis using DNA polymerase I and RNaseH. Double-stranded cDNA was end-repaired using T4 DNA polymerase, T4 polynucleotide kinase and Klenow polymerase. The DNA fragments were then adenylated using Klenow exo-polymerase to allow the ligation of Illumina Truseq HT adapters (D501–D508 and D701–D712). All enzymes were purchased from Enzymatics. Following library preparation, quality control and quantification were performed using a 2100 Bioanalyzer instrument (Agilent) and the Quant-iT PicoGreen dsDNA Reagent (Invitrogen), respectively. Libraries were sequenced using Illumina HiSeq4000 sequencers to generate 50-bp single-end reads.
7
Whole-Genome Sequencing of P. falciparum Mutants
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Genomic DNAs used for whole-genome sequencing were purified from pfcas9 parasites and pfap2g-ko parasites as described above. The obtained genomic DNA was further purified using a NucleoSpin gDNA Clean-up Kit (Macherey-Nagel). Each of the purified genomic DNA samples was sheared to an average size of 600 bp with Covaris S220 (Covaris), and then, from the sheared DNA, DNA libraries were prepared using the KAPA Hyper Prep Kit (KAPA Biosystems) and TruSeq HT adapters (Illumina) according to the manufacturer's instructions. Whole-genome sequencing was performed on the Illumina MiSeq platform (Illumina) with 251-bp and 301-bp single-end sequencing. parasite, and the mutant line, i.e., the pfap2g-ko parasite, was carried out with GenotypeGVCFs of GATK. Then, SNPs and indels were selected with standard filtering parameters. The variants that were called uniquely in the mutant line were confirmed by mapping using the genome browser IGV (http://software.broadinstitute.org/software/igv/home) to remove false-positive variants.
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