GSM3560422: respiratory burst rep1; Agaricus bisporus; RNA-Seq
Samples were crushed in LN using mortar and pestle and further ground to a fine powder for 1 min using a Tissuelyser II with a LN pre-cooled steel grinding jar (Qiagen, Venlo, Netherlands) at 30 Hz. Strand-specific mRNA-seq libraries for the Illumina platform were generated and sequenced at BaseClear BV (Leiden, The Netherlands). High-quality total RNA (checked and quantified on a Bioanalyzer (Agilent)) was used as input for the so-called dUTP library preparation method (Parkhomchuk D et al, Nucleic Acid Research 2009; Levin J et al, Nature Methods 2010). Briefly, the mRNA fraction was purified from total RNA by polyA capture, fragmented and subjected to first-strand cDNA synthesis with random hexamers in the presence of Actinomycin D. The second-strand synthesis was perform incorporating dUTP instead of dTTP. Barcoded DNA adapters were ligated to both ends of the double-stranded cDNA and subjected to a PCR amplification. The resultant library was checked on a Bioanalyzer (Agilent) and quantified. The libraries were multiplexed, clustered, and sequenced on an Illumina HiSeq 2500 (HiSeq Rapid SBS v2 chemistry) with a single-read 50 cycles sequencing protocol and indexing.
GEO Accession
GSM3560422