Two hundred microliter of lysate for each sample was purified using the RNA Clean & Conentrator-25 kit (R1018; Zymo Research, Irvine, USA) following the manufacturer’s instructions. DNA contamination was eliminated by performing the DNase I In-Column treatment (E1010; Zymo Research). Samples were eluted in 25 µl DNase/RNase-free water. The Quantseq 3′ mRNA kit (Lexogen, Vienna, Austria) was used for library preparation74 (link). Fourty eight libraries were sequenced on 4 Ion Torrent chips with an expected read yield of >60 million reads per chip. The final average coverage over the 6000 genes was more than 800 reads per gene. Adapters, sequences of less than 30 bp in length, Poly-A tails and sequences with quality score lower than 15 were removed using the program Cutadapt75 (link). Read quality statistics were retrieved from the program FastQC76 . Raw sequences can be downloaded under the NCBI bio project ID PRJNA480398.
Quantseq 3 mrna kit
Manufactured by Lexogen
Sourced in Austria, United States
The QuantSeq 3'-mRNA kit is a library preparation kit designed for 3'-end sequencing of polyadenylated (poly(A)) RNA. The kit generates libraries from total RNA samples for next-generation sequencing (NGS) on Illumina platforms. The core function of the kit is to convert poly(A) RNA into cDNA, which is then amplified and barcoded for multiplexed sequencing.
Automatically generated - may contain errors
Lab products found in correlation
Tapestation, by Agilent Technologies (3 mentions)
Nextseq 500, by Illumina (2 mentions)
Hiseq 4000, by Illumina (1 mentions)
High sensitivity dna kit, by Agilent Technologies (1 mentions)
Direct zol rna kit, by Zymo Research (1 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Bioanalyzer, by Agilent Technologies (1 mentions)
Reagent kit v3, by Illumina (1 mentions)
Miseq machine, by Illumina (1 mentions)
Purelink rna mini kit, by Thermo Fisher Scientific (1 mentions)
Rneasy kit, by Qiagen (1 mentions)
Qiashredder, by Qiagen (1 mentions)
Direct zol microprep kit, by Zymo Research (1 mentions)
6 protocols using quantseq 3 mrna kit
1
3' mRNA Sequencing Protocol for Gene Expression
2
Transcriptome Analysis of HT1 Overexpression in 293T Cells
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A total of 2.0 μg DNA (pHT1 or empty pEF.Bos vector, see above), was transfected using Lipofectamine 3000 (Invitrogen) in 8.0E+05 293T cells. After 48 hrs, transfected 293T cells and CycT1 KO 293T cells were washed using PBS, lysed using TRIzol (Invitrogen), and RNA was purified using DirectZol RNA kit (Zymo research) followed by DNase I treatment (Turbo DNA free Ambion). 500 ng RNA was used to prepare mRNA libraries using QuantSeq 3’ mRNA kit (Lexogen) for Illumina. High Sensitivity DNA kit (Agilent) was used for quality control of the libraries using a Bioanalyzer (Agilent). Libraries were sequenced using a HiSeq4000 (Illumina), and the reads were analyzed using the differential expression pipeline from BlueBee, using empty vector transfected 293T cells as a control for HT1-expressing 293T cells and for CycT1 KO 293T cells. Each condition was tested in triplicate experiments.
3
Transcriptome Sequencing and Analysis
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Libraries were prepared with the QuantSeq 3′ mRNA kit (Lexogen, Greenland, NH, USA) using 0.5 μg of total RNA purified as above. Sequencing was performed in an Illumina MiSeq machine with Reagent Kit v3 (single end, 80–125 nt/read). Two biological replicates were sequenced, obtaining a total number of 8.4–12.9 million reads per condition. Mapping of fastq files to generate SAM files was carried out with the Bowtie2 software53 (link) in local mode (95.1–97.3% mapped reads). The SAM files were analyzed with the SeqMonk software (www.bioinformatics.bbsrc.ac.uk/projects/seqmonk ). Mapped reads were counted using CDS probes (extended 100 nt downstream the open reading frame because the library is biased towards the 3′-end of mRNAs) and corrected for the largest dataset. Raw data was subjected to diverse filters to remove sequences with a low number of reads.
4
Lamin A/C Knockout RNA-seq Analysis
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Total RNA for Lamin A/C knockout experiment was isolated from 1.5 x 106 cells using using a PureLink RNA Mini Kit (ThermoFisher) according to the manufacturer’s protocol. RNA samples were submitted to the Wistar Institute genomics core facility for initial analysis of RNA quality, with each sample having a RIN value greater than 8.5 (TapeStation, Agilent Technologies). Sequencing library preparation was then completed using the QuantSeq 3’-mRNA kit (Lexogen) to generate Illumina-compatible sequencing libraries according to the manufacturer’s instructions. Single reads of 75 bp were obtained using a NextSeq 500 sequencer. RNA-seq data was aligned using bowtie2 [75 (link)] against hg19 version of the human genome and all unaligned reads were then aligned against NC_007605.1 version of EBV genome and RSEM v1.2.12 software [76 (link)] was used to estimate raw read counts and RPKM for EBV genes. DESeq2 [77 (link)] was used to estimate significance of differential expression between groups pairs. Genes that passed nominal p<0.05 (FDR<5%) threshold were reported unless stated otherwise. Datasets are available in GEO under the accession number GSE181012.
5
Isolation and RNA-seq of MUN14 and M14 Cells
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MUN14 and M14 cells obtained from the brains and kidneys of mice were filtered through a 40μM and then a 30μM filter prior to sorting for GFP to isolate MUN14 or M14 cells from the brain or kidney. Total RNA was extracted using a QiaShredder followed by RNA isolation with the RNeasy kit (Qiagen).
RNA samples were submitted to the Wistar Institute genomics core facility for initial analysis of RNA quality, with each sample having a RIN value greater than 8.5 (TapeStation, Agilent Technologies). Sequencing library preparation was then completed using the QuantSeq 3’-mRNA kit (Lexogen) to generate Illumina-compatible sequencing libraries according to the manufacturer’s instructions. Sequencing was done with an Illumina NextSeq500 on high output mode to generate ~4x108 75bp reads across 8 multiplexed and pooled samples.
RNA samples were submitted to the Wistar Institute genomics core facility for initial analysis of RNA quality, with each sample having a RIN value greater than 8.5 (TapeStation, Agilent Technologies). Sequencing library preparation was then completed using the QuantSeq 3’-mRNA kit (Lexogen) to generate Illumina-compatible sequencing libraries according to the manufacturer’s instructions. Sequencing was done with an Illumina NextSeq500 on high output mode to generate ~4x108 75bp reads across 8 multiplexed and pooled samples.
6
Bulk Transcriptome Profiling from Cells
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Total RNA was isolated from 1.5 x 106 cells using the Direct-zol microprep kit following the manufacturer’s protocol (Zymo Research). RNA samples were submitted to the Wistar Institute genomics core facility for initial analysis of RNA quality, with each sample having a RIN value greater than 8.5 (TapeStation, Agilent Technologies). Sequencing library preparation was then completed using the QuantSeq 3’-mRNA kit (Lexogen) to generate Illumina-compatible sequencing libraries according to the manufacturer’s instructions. Sequencing was done with an Illumina NextSeq500 on high output mode to generate ~4x108 1 x 75bp reads across 8 multiplexed and pooled samples.
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