Protocol detail

RNA Extraction and Sequencing Protocol

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Total RNA was extracted using the mirVana miRNA isolation kit according to the manufacturer’s protocol (Invitrogen, Waltham, MA). The RNA quality was analyzed using a 2100 Bioanalyzer Instrument (Agilent Technologies, Santa Clara, CA) and concentration was measured using a Qubit fluorometer (Life Technologies, Carlsbad, CA). For sRNA and mRNA sequencing, the total RNA was sent for library preparation and paired-end sequencing at the National Genomics Infrastructure (NGI), Stockholm, Sweden. The sRNA library was generated using TruSeq small RNA kit (Illumina, San Diego, CA), while the mRNA library was generated using a TruSeq Stranded mRNA Poly(A) selection kit (Illumina, San Diego, CA). The sRNA and mRNA libraries were sequenced on a NovaSeq SP flow cell with a 2 × 50-bp reads and NovaSeqXp workflow in S4 mode flow cell with 2 × 151 setup, respectively, using Illumina NovaSeq6000 equipment at NGI Stockholm. The Bcl to FastQ conversion was performed using bcl2fastq_v2.19.1.403 from the CASAVA software suite (126 (link)). The quality scale used was Sanger/phred33/Illumina 1.8+.

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Piombo E., Vetukuri R.R., Broberg A., Kalyandurg P.B., Kushwaha S., Funck Jensen D., Karlsson M, & Dubey M. (2021). Role of Dicer-Dependent RNA Interference in Regulating Mycoparasitic Interactions. Microbiology Spectrum, 9(2), e01099-21.

Publication 2021

mirVana miRNA isolation kit 2100 Bioanalyzer Instrument Qubit fluorometer TruSeq small RNA kit TruSeq Stranded mRNA Poly(A) selection kit NovaSeq6000

Cell Isolation Library Mirna Mrna Poly a

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Variable analysis

independent variables

Total RNA extraction using the mirVana miRNA isolation kit
RNA library preparation using TruSeq small RNA kit (for sRNA) and TruSeq Stranded mRNA Poly(A) selection kit (for mRNA)
Sequencing on Illumina NovaSeq6000 equipment

dependent variables

RNA quality analyzed using a 2100 Bioanalyzer Instrument
RNA concentration measured using a Qubit fluorometer
SRNA and mRNA sequencing data

control variables

Manufacturer's protocol for total RNA extraction and library preparation
Sanger/phred33/Illumina 1.8+ quality scale used for sequencing

controls

No positive or negative controls were explicitly mentioned in the input.

Annotations

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