Nuclei and nucleoli were isolated as previously described (Pontvianne et al. 2016a ) using a S3 cell sorter (Biorad®) at the Bioenvironment cytology platform (Perpignan University, Perpignan, France). Sorted nuclei or nucleoli were treated with RNase A and proteinase K prior to phenol/chloroform DNA purification, followed by two precipitation steps. DNA libraries were generated via the kit Nextera XT DNA sample preparation (Illumina®) according to manufacturer's instructions and were, then, subjected to high throughput paired-end 2X150nt sequencing on a Next-seq 550 apparatus (Illumina ®) at the Bioenvironment sequencing platform (Perpignan University, Perpignan, France). NADs identification was then performed as described in (Carpentier et al. 2018) (link). DNA-seq data are deposited at the ENA European Nucleotide Archive under the reference PRJEB36061.
Next seq 550 apparatus
Manufactured by Illumina
Sourced in United States
The NextSeq 550 is a next-generation sequencing system designed for a wide range of applications, including gene expression analysis, targeted resequencing, and small RNA sequencing. The system utilizes sequencing-by-synthesis technology to generate high-quality sequencing data. The NextSeq 550 is capable of producing up to 120 gigabases of data per run, with read lengths up to 150 base pairs. The system features a user-friendly interface and is compatible with a variety of library preparation kits and analysis software.
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Lab products found in correlation
2 protocols using next seq 550 apparatus
1
Nuclei and Nucleoli Isolation and Sequencing
2
RNA-seq Analysis of Triple-Negative Breast Cancer
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Using RNA extracted in 2.2.2.2., cDNA libraries were prepared with Illumina Stranded mRNA Prep, Ligation kit (Illumina, Inc., San Diego, CA, USA) according to the manufacturer’s instructions. Paired-end read (2 × 74 bp) RNA sequencing (RNAseq) was performed on a NextSeq 550 apparatus (Illumina) with a NextSeq 500/550 Mid Output Kit v2.5 (Illumina, Inc., San Diego, CA, USA). Raw FASTQ data were processed as described previously [44 (link)]. RNA expression data of MDA-MB-453, MDA-MB-231 and MFUM-BrTNBC-1 (4th passage) were compared to the MCF-7 epithelial CL.
RNA was further analysed using RNA-seq variant calling pipeline. Raw reads were aligned to reference genome GRCh38 using STAR aligner [45 (link)]. PCR duplicates were marked and sorted using PicardTools v2.26.6 (broadinstitute.github.io/picard/; accessed on 13 December 2021. Subsequently, cigar reads spanning splice sites were split, and base quality scores were recalibrated using Genome Analysis Toolkit (GATK) (v4.2.3.0, Broad Institute, Cambridge, MA, USA) [46 (link)]. Variant calling was performed using HaplotypeCaller implemented in GATK with a minimum phred scaled confidence threshold of 20.
RNA was further analysed using RNA-seq variant calling pipeline. Raw reads were aligned to reference genome GRCh38 using STAR aligner [45 (link)]. PCR duplicates were marked and sorted using PicardTools v2.26.6 (broadinstitute.github.io/picard/; accessed on 13 December 2021. Subsequently, cigar reads spanning splice sites were split, and base quality scores were recalibrated using Genome Analysis Toolkit (GATK) (v4.2.3.0, Broad Institute, Cambridge, MA, USA) [46 (link)]. Variant calling was performed using HaplotypeCaller implemented in GATK with a minimum phred scaled confidence threshold of 20.
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