The Illumina paired-end libraries (Illumina, Inc.) techniques were used to detect and design the entire genome library and sequencing, as previously explained [30 (link)]. The amplified DNA (900 ng to 1 μg) from the advanced melanoma cell lines C8161, MUM-2B, and SK-MEL-28 was sheared into fragments of about 300 bp using a Covaris E220 system(Covaris Inc.). An aliquot of adaptor-ligated DNA was generated to use in the enrichment qPCR to establish the adaptor for Illumina PE sequencing. The pooled DNA (600 ng) from four barcoded libraries was utilized for hybridization and post-hybridization amplification according to the manufacturer’s instructions (SureSelectXT Target Enrichment System for Illumina Paired-End Sequencing Library; Illumina, Inc.). The results of the PCR amplification of cell lines were quality confirmed, and Illumina HiSeq 2000 (Illumina, Inc.) was used for WGS, with an average coverage depth of between 10x and 30x.
Sureselectxt target enrichment system for paired end sequencing library
Manufactured by Illumina
Sourced in United States
The SureSelectXT Target Enrichment System is a laboratory equipment product from Illumina designed for Paired-End Sequencing Library preparation. The core function of this system is to selectively capture and enrich specific target sequences from a DNA sample, enabling targeted sequencing analysis.
Automatically generated - may contain errors
Lab products found in correlation
Hiseq 2000, by Illumina (3 mentions)
Paired end libraries, by Illumina (2 mentions)
E220 system, by Covaris (1 mentions)
Earray platform, by Agilent Technologies (1 mentions)
Sureselect human all exon v5 kit, by Agilent Technologies (1 mentions)
Bioruptor, by Diagenode (1 mentions)
Truseq small rna sample preparation protocol, by Illumina (1 mentions)
Hiscansq sequencer, by Illumina (1 mentions)
6 protocols using sureselectxt target enrichment system for paired end sequencing library
1
Whole Genome Sequencing of Melanoma Cell Lines
2
Targeted Sequencing of Genomic Regions
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Probes were designed to capture open reading frame (ORF), 3′UTR, 5′UTR, and 2KB of upstream promotor of selected genes on Agilent eArray platform against hg19 human genome. Genomic DNA from patients were sonicated into fragments. A DNA library from an individual was barcoded and used for generating the library for paired-end sequencing according to manufacturers' instruction for SureSelect XT Target Enrichment System for Illumina Paired-End Sequencing Library (Agilent). Reads were aligned in the reference genome with Burrows–Wheeler Aligner after filtering low quality reads. Single-nucleotide variants were then identified according to a previous report (15 (link)).
3
Targeted Exome Sequencing for Biomedical Research
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To prepare NGS libraries, we followed the SureSelectXT Target Enrichment System for Illumina Paired-End Sequencing Library (Agilent) protocol specifications starting from 3 μg of pooled DNA. DNA was sheared by using Bioruptor (Diagenode) to obtain fragments of ~250 bp. Exome capture was performed by using a SureSelect Human All-Exon V5 kit (Agilent), which targets ~50 Mb of coding regions of human genes. Hybridization and capture were performed separately for each sample library prepared. Sequencing was performed on Illumina’s HiSeq 2000 at IGA Technology Services (Udine, Italy) using a paired-end approach with runs of 100 bp.
4
Whole Genome Sequencing of Circulating Tumor Cells
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The design of whole genome library and sequencing were detected using Illumina paired-end libraries (Illumina, Inc.) methods as previously described (28 (link)). Briefly, 900 ng to 1 µg of extracted DNA from the amplification products of CTCs by MALBAC or from tumor tissues was sheared into fragments of approximately 300 bp using a Covaris E220 system (Covaris Inc.). The adaptor-ligated DNA was prepared, and enrichment q;PCR was performed on an aliquot of adaptor-ligated DNA to complete the adaptor for Illumina PE sequencing. A total of 600 ng of pooled-DNA from four barcoded libraries was used for hybridization and post-hybridization amplification following the manufacturer's protocol (SureSelectXT Target Enrichment System for Illumina Paired-End Sequencing Library; Illumina, Inc.). Then, the result of the PCR amplification of single CTCs were quality checked and WGS was performed with the Illumina HiSeq 2000 (Illumina, Inc.). to achieve an average of ~10× and ~30× coverage depth, respectively.
5
RNA-seq Analysis of SH-SY5Y Cell Differentiation
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Total RNA was extracted from SH-SY5Y differentiated and undifferentiated cells and 1 μg from each extraction was used to prepare indexed libraries according to the Illumina TruSeq Small RNA Sample Preparation protocol with the following modifications: after 5′ Adapter ligation, two aliquots from each ligation reaction product were taken to obtain a total of four indexed libraries; and a total of 15 PCR cycles were performed. The four libraries were concentrated to approximately 30 ng/μl with a vacuum concentrator. Next, the two differentiated libraries were pooled together as well as the two undifferentiated libraries. From each pool, 100 ng were used for hybridization with a custom SureSelect sRNA Target Enrichment library following Agilent’s SureSelectXT Target Enrichment System for Illumina Paired-End Sequencing Library (version 1.3.1 [61 ]), replacing the SureSelect Block mix supplied in the kit with a custom Block mix provided by Agilent. Post-capture PCR amplification was performed using primers complementary to the Illumina P5 and P7 adapter region sequences. Sequencing and data pre-processing were performed as described above.
6
Exome Sequencing Protocol for Comparative Genomic Analysis
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The genomic DNAs of patients no. 6 and 10 (tumor and para-carcinoma tissue), as presented in Table I , were randomly fragmented into 150–200 bp. Next, library construction, exome capture and sequencing were performed according to the protocol of the Sure SelectXT Target Enrichment System for Illumina Paired-End Sequencing Library, Illumina HiSeq and MiSeq Multiplexed Sequencing Platforms (Illumina, Inc., San Diego, CA, USA). Sequencing by synthesis was performed on a HiScanSQ sequencer (Illumina, Inc.) using the paired-end method. BAM files summarizing Burrows-Wheeler alignments that were mapped to the hg19 reference sequence were generated using the Genome Analysis Toolkit (GATK) (22 (link),23 ). The raw reads with all characteristics that were not confidently aligned, exhibited a ratio of N >10%, a length <25 bp without adapter and a quality score of <20 by Phred-scaled mapping were excluded from further analysis. The single nucleotide variants (SNVs), insertions and deletions were detected and annotated by the GATK and Annovar software (release date Feburay 1st, 2016; http://www.openbioinformatics.org/annovar/ ) (24 (link)).
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