Total RNA was extracted using the RNeasy Plus Mini Kit following manufacturer’s instructions. RNA quality was confirmed using the Bioanalyzer RNA 600 Nano kit (Agilent). Libraries were generated with 200ng of RNA input using the KAPA mRNA HyperPrep kit and KAPA SeqCap adapters. Fragmentation was carried out at 94°C for 6 minutes and libraries amplified for 12 cycles. Pooling ratios for multiplex library sequencing was calculated according to individual library concentrations and size distributions assessed with the Qubit dsDNA high-sensitivity and Agilent Bioanalyzer DNA 1000 assays, respectively. Pooled libraries were diluted and processed for sequencing using the Illumina HiSeq 2500 platform.
Bioanalyzer rna nano 600 kit
Manufactured by Agilent Technologies
Sourced in United States
The BioAnalyzer RNA Nano 600 kit is a lab equipment product designed for the analysis of RNA samples. The kit provides the necessary components to perform RNA quality assessment and quantification using the Agilent BioAnalyzer platform.
Automatically generated - may contain errors
Lab products found in correlation
Quantseq 3 mrna seq library prep kit fwd for kit, by Illumina (3 mentions)
Nextseq 500 instrument, by Illumina (3 mentions)
Total rna extraction kit, by Norgen Biotek (2 mentions)
Tryple, by Thermo Fisher Scientific (2 mentions)
Gemcitabine, by Merck Group (2 mentions)
Bluebee integrated, by Lexogen (2 mentions)
Bioanalyzer dna 1000, by Agilent Technologies (1 mentions)
Hiseq 2500 platform, by Illumina (1 mentions)
Truseq stranded total rna sample preparation protocol, by Illumina (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
5 protocols using bioanalyzer rna nano 600 kit
1
Illumina RNA-Seq Library Preparation
2
RNA Isolation and Sequencing Workflow
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RNA was isolated with TRIzol reagent (Invitrogen, Thermo Fisher Scientific Inc.) according to manufacturer's instructions. RNA quality was assessed by Agilent Bioanalyzer RNA 600 Nano Kit and RIN values >9 were accepted. Random fragmentation, cDNA synthesis and library generation were performed according to TruSeq Stranded Total RNA Sample Preparation protocol (Illumina). Libraries were subjected to sequencing, mapped onto the human reference genome and the resulting unique reads were counted across annotated protein-coding transcripts. For details, see Supporting File 1: Supporting Methods .
3
Gemcitabine Treatment on Cell Line
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Cells were seeded at a density of 0.6×10^6 cells per well of a 6-well plate in triplicate. The following day media was removed and 3ml of media per well with or without 1.5nM of gemcitabine (Sigma #G6423) was added and incubated for 24 hours. Media was removed, cells were washed with PBS (Gibco #10010072), and released from the plate with 2 ml of TrypLE (Gibco #12604-021) per well. After centrifugation, pellets were frozen at −80C until RNA extraction. For RNA extraction, of cell pellets 350 ul of RL Buffer plus 1% BME from the Norgen Total RNA extraction kit and extraction proceeded per manufacturer’s instructions including use of the DNase kit (Norgen # 37500, 25720). RNA quality was verified with the Agilent BioAnalyzer RNA Nano 600 kit (cat# 5067 - 1512) with the RIN range between 9.2–10. RNA-sequencing libraries were made using Lexogen QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina kit (cat# 015.24) with 250 ng of RNA input. They were pooled and sequenced on an Illumina NextSeq 500 instrument with 75 bp single-end reads. Read counts averaged 4 million reads and average percent over Q30 bases of 93.65%. Lexogen’s BlueBee integrated QuantSeq data analyses pipeline was used for trimming, mapping, and alignment and DESeq2 was used for differential expression [34 (link)]. Heatmaps were generated using iDEP.95 [35 (link)].
4
RNA-sequencing of Cell Pellets
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RNA from cell pellets was extracted using Norgen Total RNA following the manufacturer’s protocol (Thorold, ON, USA, cat# 37500, 25710). RNA quality was verified with the Agilent BioAnalyzer RNA Nano 600 kit (Santa Clara, CA, USA, cat# 5067-1512) with the RIN range between 9–10. RNA-sequencing libraries were made using Lexogen QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina kit (Vienna, Austria, cat# 015.24) with 250 ng of RNA input. They were pooled and sequenced on an Illumina NextSeq 500 instrument with 75 bp single end reads. Read counts averaged 11.2 million reads and average Q30% was 94.28. Lexogen’s BlueBee integrated QuantSeq data analyses pipeline was used for trimming, mapping, and alignment, and DESeq2 implemented in R was used for determination of differential expression [26 (link)]. All differentially expressed genes with a base mean > 5, log2 fold change of < −0.5 or > 0.5, and p value < 0.2 (unadjusted) were analyzed using WebGestalt (http://www.webgestalt.org ) (accessed on 4 December 2021) [25 (link)] for gene set enrichment analysis and Qiagen Ingenuity Pathway Analysis (https://digitalinsights.qiagen.com/IPA ) (accessed on 18 February 2022) [27 (link)]. Data for RNA-sequencing analysis can be found on the Gene Expression Omnibus with the accession number GSE179087.
5
Quantifying RNA Expression Changes in Gemcitabine-Treated Cells
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Cells were seeded at a density of 0.6 × 10^6 cells per well of a 6-well plate in triplicate. The following day media was removed and 3ml of media per well with or without 1.5nM of gemcitabine (Sigma #G6423) was added and incubated for 24 hours. Media was removed, cells were washed with PBS (Gibco #10010072), and released from the plate with 2 ml of TrypLE (Gibco #12604-021) per well. After centrifugation, pellets were frozen at -80 C until RNA extraction. For RNA extraction, of cell pellets 350 ul of RL Buffer plus 1% BME from the Norgen Total RNA extraction kit and extraction proceeded per manufacturer’s instructions including use of the DNase kit (Norgen # 37500, 25720). RNA quality was verified with the Agilent BioAnalyzer RNA Nano 600 kit (cat# 5067 − 1512) with the RIN range between 9.2–10. RNA-sequencing libraries were made using Lexogen QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina kit (cat# 015.24) with 250 ng of RNA input. They were pooled and sequenced on an Illumina NextSeq 500 instrument with 75 bp single-end reads. Read counts averaged 4 million reads and 93.65% of bases exceeding Q30. Lexogen’s BlueBee integrated QuantSeq data analyses pipeline was used for trimming, mapping, and alignment and DESeq2 was used for differential expression [23 ]. Heatmaps were generated using iDEP.95 [24 ].
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