Total RNA was isolated from cells using the phenol–chloroform extraction method and treated with DNase I (Ambion). RNA was depleted of rRNA with the RiboCop rRNA Depletion Kit (Lexogen) and used for library preparation using the SENSE Total RNA-seq Library Prep Kit (Lexogen). For the sequencing of the dsRNA, immunoprecipitation was performed with J2 (dsRNA-specific antibody), then 1 µg per sample was depleted of rRNA with RiboCop rRNA Depletion Kit (Lexogen). The rRNA depleted dsRNA was used for library preparation with SENSE Total RNA-seq Library Prep Kit (Lexogen). The libraries were prepared with TruSeq Ilumina adapters. Sequencing was performed on the NextSeq 500/550 sequencer.
Sense total rna seq library prep kit
Manufactured by Lexogen
Sourced in Austria
The SENSE Total RNA-Seq Library Prep Kit is a laboratory equipment product designed for the preparation of RNA sequencing libraries. It provides a method for converting total RNA into a sequencing-ready library for subsequent analysis.
Automatically generated - may contain errors
Lab products found in correlation
Hiseq 2500, by Illumina (3 mentions)
Nextseq 500 sequencer, by Illumina (3 mentions)
Nextseq 500, by Illumina (3 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (3 mentions)
Trizol, by Thermo Fisher Scientific (3 mentions)
Ribo zero magnetic kit, by Illumina (2 mentions)
Agilent rna 6000 nano kit, by Agilent Technologies (2 mentions)
Ribocop rrna depletion kit, by Lexogen (2 mentions)
Kapa library quant kit rt qpcr, by Roche (2 mentions)
Bioanalyzer rna nano chip, by Agilent Technologies (2 mentions)
Quantseq 3 mrna seq library prep kit, by Lexogen (2 mentions)
Ribocop rrna depletion kit v1, by Lexogen (2 mentions)
Nextseq 500 550 system, by Illumina (2 mentions)
Nextseq 500 550 high output kit v2 75 cycle, by Illumina (2 mentions)
Bcl2fastq2, by Illumina (1 mentions)
Qubit dsdna hs assay kit, by Thermo Fisher Scientific (1 mentions)
Hiseq rapid sbs kit, by Illumina (1 mentions)
Qiashredder, by Qiagen (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Turbo dnase, by Thermo Fisher Scientific (1 mentions)
20 protocols using sense total rna seq library prep kit
1
Total RNA Isolation and dsRNA Sequencing
2
Comprehensive RNA-seq Library Preparation
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For RNA-sequencing, rRNA was removed from total RNAs (each 5 ug) by using Ribo-Zero Magnetic kit (epicentre, Inc., Madison, WI, USA). Genomic libraries were constructed using SENSE Total RNA-Seq Library Prep Kit (Lexogen, Inc., Vienna, Austria) according to the manufacturer’s protocol. The starter/stopper heterodimers, containing Illumina-compatible linker sequences, were randomly hybridized to remained RNA, initiating library production. The starter was extended to the next hybridized heterodimer by a single-tube reverse transcription and ligation reaction, followed by the stopper ligated with newly-synthesized cDNA inserts. The resulting library was amplified, following second strand synthesis to release the library from beads, thus introducing barcodes. As paired-end 100 sequencing, the sequencing was performed by HiSeq 2500 (Illumina, Inc., San Diego, CA, USA). The FPKM (fragments per kilobase of exon per million fragments) was used for determining the expression levels of gene regions.
3
RNA-Seq Analysis of SNCA and SNCA-AS1 in SH-SY5Y Cells
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500 ng of total RNA from SH‐SY5Y, SH‐SY5Y‐SNCA‐AS1, and SH‐SY5Y‐SNCA was used to prepare libraries with the SENSE Total RNA‐Seq Library Prep Kit (Lexogen) and sequenced by Illumina NextSeq 500 sequencer. Qualities of sequencing libraries were assessed by 2100 Bioanalyzer with DNA1000 assay (Agilent) and quantified with Qubit dsDNA HS Assay Kit (Invitrogen). FastQ files were generated via Illumina bcl2fastq2 starting from raw sequencing reads produced by Illumina NextSeq 500 sequencer (Version 2.17.1.14‐ http://support.illumina.com/downloads/bcl‐2fastq‐conversion‐software‐v217.html ). The raw data obtained from the RNA‐Seq analysis are deposited on the Gene Expression Omnibus repository: GSE183410 for SH‐SY5Y and GSE186255 for SH‐SY5Y‐SNCA and SH‐SY5Y‐SNCA‐AS1. Gene and transcript intensities were computed using STAR/RSEM software (Li & Dewey, 2011 (link)) using Gencode Release 27 (GRCh38) as a reference, using the “stranded” option. Differential expression analysis was performed using R package DESeq.2 (Love et al., 2014 (link)). Coding and non‐coding genes were considered differentially expressed and retained for further analysis with |log2(SH‐SY5Y‐SNCA‐AS1/SH‐SY5Y)| ≥1 and a FDR ≤0.1 and |log2(SH‐SY5Y‐SNCA/ SH‐SY5Y)| ≥1 and a FDR ≤0.1.
4
RNA Extraction and Sequencing Protocol
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After the drug incubations, RNA from 1 ml of cell suspension of each sample was extracted using the RNeasy Mini Kit (Qiagen) and QIAshredder (Qiagen) according to the manufacturer’s protocol. Ribosomal RNA was depleted using RiboCop rRNA Depletion Kit version 1.2 (Lexogen), and sequencing libraries were prepared with SENSE Total RNA-Seq Library Prep Kit (Lexogen) following the manufacturer’s protocol. Libraries were sequenced at Science for Life Laboratory (SciLifeLab, Stockholm, Sweden) using the HiSeq 2500 (Illumina) with HiSeq Rapid SBS Kit v2 chemistry and a 1 × 51 setup.
5
Transcriptomic Analysis of Neurodegenerative Disorders
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Frozen slices from four brain areas (PL, BG, HiC, and SN) of the two kindred cases and of the nondemented control were used to perform transcriptome analysis. Total RNA was isolated using Trizol® (Life Science Technologies, Leawood, KS, USA), and RNA quality and integrity were evaluated based on the RNA Integrity Number (RIN), acquired using the Agilent 2100 Bioanalyzer (Agilent RNA 6000 Nano Kit). RNA libraries were prepared in duplicate using the SENSE Total RNA-Seq Library Prep Kit (Lexogen, Vienna, Austria) according to the manufacturer’s protocol. Libraries were sequenced using NextSeq 500 Sequencer (Illumina, San Diego, CA, USA). FastQ files generation and transcript mapping and quantification were performed as described in Zucca et al. 2019 [40 (link)]. Differential expression analysis with respect to the healthy control was performed using R package EBSeq2 [41 (link)] and retained for further analysis with |log2(disease sample/healthy control)| ≥ 1, and an FDR ≤ 0.1. qPCR was performed to validate RNA-seq data. RNA dataset generated during the current study is available in the GEO repository (Reference number GSE193438).
6
Purifying RNA Samples for Sequencing
7
RNA-seq Library Preparation from Sorted SSCs
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RNA was isolated from sorted SSCs by using QIAzol Lysis Reagent (79306, Qiagen) and following the manufacturer’s instructions. To generate RNA-seq libraries total RNA was treated with DNAse I, in 10X Buffer (AMPD1, Sigma). RNA was purified using Rneasy MinElute columns (74204, Qiagen) and ribosomal RNA depletion was performed with the RiboCop kit (Lexogen). Ribo-depleted RNA was used to generate libraries with the SENSE Total RNA-Seq Library Prep Kit (Lexogen) following the manufacturer’s instructions. Libraries were sequenced with an Illumina HiSeq2500 on a 50 bp, single-end run.
8
Total RNA and 3'end sequencing of KD cell lines
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For total RNA sequencing of HEK293T, RNA from SFPQ, NONO, and CTRL KD (using two different siRNAs for each condition with biological duplicates) were rRNA depleted using RiboCop rRNA Depletion Kit V1.2 (Lexogen) according to manufacturer’s protocol. Subsequent cDNA libraries were prepared using SENSE Total RNA-Seq Library Prep Kit (Lexogen) following manufacturer’s protocol.
For 3’end sequencing, cDNA libraries from HEK293T were prepared using QuantSeq 3' mRNA-Seq Library Prep Kit (Lexogen). For both methods, RNA quality was determined using the BioAnalyzer RNA nanochip (Agilent) and library concentration was quantified with KAPA Library Quant KIT RT-qPCR (Roche). Total RNAseq was done as 100nt paired-end sequencing and performed using the Illumnia platform (HiSEQ4000, BGI, Copenhagen), while for 3’end sequencing, 75nt single-end sequencing was performed at MOMA (Aarhus University Hospital) on a NextSeq500. For total RNA sequencing of HepG2 cells, library preparation and sequencing was performed at BGI (Copenhagen) using BGIseq.
For 3’end sequencing, cDNA libraries from HEK293T were prepared using QuantSeq 3' mRNA-Seq Library Prep Kit (Lexogen). For both methods, RNA quality was determined using the BioAnalyzer RNA nanochip (Agilent) and library concentration was quantified with KAPA Library Quant KIT RT-qPCR (Roche). Total RNAseq was done as 100nt paired-end sequencing and performed using the Illumnia platform (HiSEQ4000, BGI, Copenhagen), while for 3’end sequencing, 75nt single-end sequencing was performed at MOMA (Aarhus University Hospital) on a NextSeq500. For total RNA sequencing of HepG2 cells, library preparation and sequencing was performed at BGI (Copenhagen) using BGIseq.
9
RNA-seq Library Preparation and Sequencing
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Total RNA was extracted from the developed stable cells as described above. The mRNA was selected using the Dynabeads mRNA DIRECT purification kit (Thermo Fisher Scientific). RNA-seq libraries were prepared using the SENSE Total RNA-Seq Library Prep kit (Lexogen, Campus-Vienna-Biocenter 5, Wien, Austria) and the libraries were amplified by a 17-cycle PCR. The quality and concentration of the prepared libraries were evaluated using TapeStation 2200 (Agilent Technologies). Equimolar amounts from each library were pooled and sent for sequencing at the SNP&SEQ platform (Uppsala University, Uppsala, Sweden) using the NovaSeq 6000 system (Illumina). A total of eight libraries, comprising one library per clone for each of the Igf2+/+_miR483+/+, Igf2dGGCT_miR483+/+, Igf2+/+_miR483−/− and Igf2dGGCT_miR483−/− cell lines, were sequenced which generated 35 to 50 million 50-bp paired-end reads per library.
10
RNA-Seq Analysis of Differential Expression
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RNA was prepared using Trizol (Life Technologies) following the manufacturer's instruction. RNA integrity was assessed with the Agilent Bioanalyzer 2100. Total RNA was subjected to library preparation (SENSE Total RNA-Seq Library Prep Kit, Lexogen) and RNA-sequencing on an Illumina HighSeq-2000 platform (SR 50 bp; > 25 Mio reads/sample). Sequence images were transformed with the Illumina software BaseCaller to bcl files, which were demultiplexed to fastq files with CASAVA (v1.8.2). The read counts of genes and transcripts were measured by Salmon (v0.14.1). The counts were normalized (TMM) using edgeR (v3.28.1) and the differential expression was performed using limma (v3.42.2, voom with quality weights). Gene ontology (GO) analysis was performed for those differentially expressed genes (adj p-value < 0.1) using topGO (v2.38.1).
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