Libraries were prepared using the Illumina TruSeq RNA Access Library kit and sequenced on a NextSeq 500 following the manufacturer's instructions. Sequenced segments were aligned with STAR2 (12 (link)) to the Human GENCODE reference (13 (link)). Gene counts were assessed using ht-seq counts (14 (link)). Differential expression was calculated using DESeq2 which is designed to analyze digital count data generated by sequencing (15 (link)). Log fold changes were shrunk using apeglm (16 (link)). Pathway enrichment analysis was done using GAGE (17 (link)).
Truseq rna access library kit
Manufactured by Illumina
Sourced in United States
The TruSeq RNA Access Library Kit is a laboratory equipment product designed for preparing RNA sequencing libraries. It enables the extraction and purification of RNA from samples, and the subsequent construction of libraries suitable for next-generation sequencing platforms.
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Lab products found in correlation
Hiseq 2500, by Illumina (2 mentions)
Hiseq 2500 sequencing system, by Illumina (2 mentions)
Nextseq 500, by Illumina (1 mentions)
Hiseq 1000, by Illumina (1 mentions)
Miseq platform, by Illumina (1 mentions)
Kingfisher flex, by Thermo Fisher Scientific (1 mentions)
Quantifluor dsdna system, by Promega (1 mentions)
Agilent 2200 tapestation, by Agilent Technologies (1 mentions)
Nextseq 500 system, by Illumina (1 mentions)
Clc genomics workbench, by Qiagen (1 mentions)
Nucleospin totalrna ffpe xs kit, by Macherey-Nagel (1 mentions)
Rna nano 6000 assay kit, by Agilent Technologies (1 mentions)
Agilent bioanalyzer 2100 system, by Agilent Technologies (1 mentions)
Nanophotometer, by Implen (1 mentions)
Truseq small rna library preparation kit, by Illumina (1 mentions)
Anti cd31, by Miltenyi Biotec (1 mentions)
Mab3715, by R&D Systems (1 mentions)
Anti epcam, by R&D Systems (1 mentions)
Facsaria 3, by BD (1 mentions)
Anti cd45, by BD (1 mentions)
7 protocols using truseq rna access library kit
1
Illumina-based RNA-seq Analysis Pipeline
2
KING-REX Transcriptome Sequencing Protocol
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KING-REX library preparation was performed according to the manufacturer’s protocol [33 ]. Libraries were sequenced in single-end on a MiSeq platform (Illumina, San Diego, CA, USA). Whole transcriptome sequencing library preparation was performed using the TruSeq RNA Access Library Kit (Illumina, San Diego, CA, USA) according to the manufacturer’s protocol and sequenced on a HiSeq1000 (Illumina, San Diego, CA, USA).
3
RNA-seq Analysis of TNBC Samples
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RNA-sequencing was performed on 60 formalin-fixed paraffin-embedded TNBC pre-treatment samples for which HER3 and EGFR scores were available (full-face slides), which were taken from patients in the Nottingham cohort who were eventually treated with adjuvant CMF chemotherapy. Cases were selected based on which had sufficient material remaining for RNA-seq. RNA-seq data were deposited in ArrayExpress (accession: E-MTAB-6729), where detailed methods can be found. In brief, samples were processed by the Emory Integrated Genomics Core using the Mag-Bind XP FFPE RNA isolation kit (Omega), KingFisher Flex magnetic particle separator (ThermoFisher), TruSeq RNA Access library kit (Illumina), Agilent 2200 TapeStation (Agilent Technologies), the QuantiFluor dsDNA System (Promega), and the Agilent High Sensitivity D1000 ScreenTape on the Agilent 2200 TapeStation (Agilent Technologies), and the HiSeq2500 (Illumina, Inc.) per the manufacturers’ instructions.
4
CTC RNA Sequencing Protocol
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A total of 40,000 CTCs were sorted from DLA product fixed with 4% paraformaldehyde. RNA sequencing was performed in duplicates using the TruSeq RNA Access Library Kit (Illumina). Libraries were sequenced on a NextSeq 500 system (Illumina). Data were analyzed and TPM values were determined using the CLC Genomics Workbench (Qiagen). Reads were mapped by applying the following parameters: mismatch cost: 2; insertion/deletion cost: 3; length fraction: 0.8; similarity fraction: 0.8; global alignment: no; strand specific: both; maximum number of hits per read: 5.
5
Transcriptome Analysis of Lung Cancer
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Based on the area of tumour tissues in the HE-stained section, total RNA was isolated from formalin-fixed paraffin-embedded tumour tissues of each lung cancer case (F1–16 and C1–16) and subjected to transcriptome analysis by RNA sequencing. In the H-FLAC cases (F1–16), tissues from only the H-FLAC components were manually obtained through macrodissection and used for the analysis. Total RNA was extracted using a NucleoSpin totalRNA FFPE XS kit (Macherey-Nagel GmbH & Co. KG, Germany), according to the manufacturer’s instructions. The purity of the isolated RNA was evaluated with optical density ratios (A260/A280 and A260/A230), using the NanoPhotometer (Implen, Munich, Germany). The RNA integrity was assessed with RNA integrity numbers (RIN) and DV200 using the RNA Nano 6000 Assay Kit and Agilent Bioanalyzer 2100 system (Agilent Technologies, Santa Clara, CA, USA). Library synthesis was performed on samples with DV200 greater than 20%. The RNA sequencing library was constructed using the TruSeq RNA Access Library Kit (Illumina, San Diego, CA, USA), followed by sequencing using the HiSeq2500 sequencing system (Illumina).
6
RNA Sequencing Library Preparation Protocols
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RNA sequencing libraries were prepared using the TruSeq RNA Access library kit (Illumina, Inc., San Diego, CA, USA) for mRNA sequencing (n = all 26 patients) or the TruSeq Small RNA Library Preparation Kits (Illumina Inc, San Diego, CA) for miRNA sequencing (subgroup of n = 12 patients, as shown in Table 1 ) according to the manufacturer`s protocol. RNA sequencing was performed on a HiSeq2500 instrument (Illumina, San Diego, CA) according to the manufacturer's protocol and as described previously (Eikrem et al. 2016a ,2016b ).
Sequencing data are available via Gene Expression Omnibus,https://www.ncbi.nlm.nih.gov/geo/ ; GSE76207 and GSE82122.
Sequencing data are available via Gene Expression Omnibus,
7
Isolation and RNA-seq of Tumor Cells
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Primary tumor tissues were minced, and single-cell suspensions were obtained. A detailed procedure is provided in the supplementary methods. The collected cells were incubated with antibody for 30 min on ice as follows: 10 μL anti-EpCAM (R&D Systems, FAB9601F) per 106 cells in 100 μL buffer; 5 μL anti-CD45 (BD, 557748) per 106 cells in 100 μL buffer; 10 μL anti-CD31 (Miltenyi Biotec, 130-092-652) per 107 cells in 100 μL buffer; and 2.5 μg of FAP (R&D systems, MAB3715) per 106 cells in 100 μL buffer. After washing with Hank’s balanced salt solution (Lonza) twice, the cells were incubated with mouse IgG (H + L) PE as follows: 10 μL per 106 cells in 100 μL buffer for 30 min on ice. After washing away the unstained secondary antibody, the cells were resuspended in 1 mL PBS and then sorted using a BD FACSARIA III (BD Biosciences). Total RNA sequencing libraries were prepared according to the manufacturer’s instructions (Illumina TruSeq RNA Access Library kit). The flow cell was then loaded on a HiSeq 2500 sequencing system (Illumina), and sequencing was performed using 2 × 100 bp read lengths.
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