Total RNA was extracted from treatment groups of adult T. castaneum using the RNeasy plus mini kit with on-column DNase treatment (Qiagen, Valencia, CA, USA), and mRNA was extracted from 5 μg total RNA using Dynabeads® mRNA DIRECT™ Micro Kit. RNA integrity of total RNA, mRNA, and cDNA was validated by TapeStation (Agilent Technologies, Santa Clara, CA USA), and quantitation was with a nanophotometer (Implen, Westlake Village, CA USA). Libraries were prepared with the Ion Total RNA-Seq Kit v2 and were individually barcoded (Ion Xpress™ RNA-Seq Barcode 1–16 Kit, Life Technologies, Carlsbad, CA, USA). Templates were prepared (Ion PITM™ Template OT2 200 Kit) and sequenced (Ion PITM™ Sequencing 200 Kit) on Ion Proton™ PITM Chips on the Ion Torrent Personal Genome Machine (Thermo Fisher Scientific, Grand Island, NY). Each chip was run with 4 barcoded samples. The distribution of reads was relatively equal among samples (Additional file 9 ). Reads were submitted to the NCBI Sequence Read Archive, accession SRP064827.
Column dnase treatment
Manufactured by Qiagen
Sourced in United States, Germany
The column DNase treatment is a laboratory equipment product that is used to remove DNA from RNA samples. It functions by using a DNase enzyme to break down any DNA present in the sample, ensuring that the final RNA preparation is free of DNA contamination.
Automatically generated - may contain errors
Lab products found in correlation
Trizol, by Thermo Fisher Scientific (2 mentions)
Ion total rna seq kit v2, by Thermo Fisher Scientific (1 mentions)
Ion torrent personal genome machine, by Thermo Fisher Scientific (1 mentions)
Ion xpress rna seq barcode 1 16 kit, by Thermo Fisher Scientific (1 mentions)
Tapestation, by Agilent Technologies (1 mentions)
Nanophotometer, by Implen (1 mentions)
Rna mini kit, by Thermo Fisher Scientific (1 mentions)
Sybr green master mix, by Quantabio (1 mentions)
Cfx96 qpcr machine, by Bio-Rad (1 mentions)
Kapa library quantification, by Roche (1 mentions)
Rneasy plant mini kit, by Qiagen (1 mentions)
3 protocols using column dnase treatment
1
Total RNA Extraction and Sequencing of Tribolium castaneum
2
Quantitative PCR Analysis of mRNA Expression
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TRIzol (Thermo Fisher Scientific, catalog no. 15596026) was used to collect mRNA. mRNA was extracted using the RNA Mini Kit (Thermo Fisher Scientific, catalog no. 12183018A) with column DNase treatment (Qiagen, catalog no. 79254). After elution, mRNA concentrations were measured using a NanoDrop and normalized to approximately 125 ng/μl. Reverse transcription was performed using SSIV VILO mater mix (Thermo Fisher Scientific, catalog no. 11756050). The cDNA was diluted 20-fold in ddH2O and mixed with SYBR Green master mix (QuantaBio, catalog no. 95073-05K) and designated qPCR primers. Each reaction was 10 μl and each test was performed in triplicate. β-actin was used as the control gene and untreated fibroblasts were used as control samples. The delta-delta CT method was used for data analysis and Log2 fold change (Log2FC) was used for data presentation.
3
Frond Tissue RNA-seq Protocol
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The sampling of frond tissue for RNA-seq was performed by snap-freezing single fronds in liquid nitrogen from specific pretreatment+treatment combinations at 0 or 60 minutes (Fig. 2A). Three biological replicates were harvested for each pretreatment+treatment combination, taken from three independently-performed assays. RNA was extracted using the RNeasy Plant mini kit with on-column DNase treatment (QIAgen, Hilden, Germany). RNA quality was validated by Agilent 2100 Bioanalyser RNA 6000 Pico assay (Agilent, Santa Clara, USA). Libraries were prepared from single frond samples using 1µg starting RNA using the TruSeq RNA library prep kit V2 (Illumina Systems, San Diego, USA) and quantified by KAPA library quantification (Roche, Basel, Switzerland) on a CFX96 qPCR machine (Bio-Rad, Hercules, USA). Paired-end sequencing was performed by HiSeq2000 sequencing system using a NextSeq 500/550 High Output kit (150 cycles) (Illumina Systems, San Diego, USA).
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