Total RNA samples (2 µg) were arrayed into 96-well plates, and polyadenylated mRNA was purified with a MultiMACS mRNA isolation kit as per the manufacturer’s instructions. First-strand cDNA was synthesized using a SuperScript cDNA Synthesis kit with random hexamer primers. The SuperScript cDNA Synthesis protocol was used for second-strand cDNA synthesis. dTTP was replaced with dUTP in the dNTP mix which allowed the second strand to be digested with UNG (Uracil-N-Glycosylase, Life Technologies, USA) in the post-adapter ligation reaction. The cDNA was quantified and checked for quality before fragmentation. Plate-based libraries were created following the BC Cancer Agency’s Michael Smith Genome Sciences Centre (BCGSC) paired-end (PE) protocol36 (link). The libraries were sequenced using Illumina HiSeq 2000 or 2500, 2 × 100 PE lanes, with v3 chemistry and HiSeq Control Software version 2.0.10.
Cancer Cdna Dttp DutpGenomeIsolationLigation Mrna Polyadenylated mrna Primers Synthesis Uracil n glycosylase
Corresponding Organization :
Other organizations :
Hospital for Sick Children, University of Calgary, McGill Genome Centre, McGill University, University of Toronto, Canada's Michael Smith Genome Sciences Centre, BC Cancer Agency, Ontario Institute for Cancer Research, University of California, San Francisco, SickKids Foundation, Institut Curie, Université Paris Cité, Inserm, Centre National de la Recherche Scientifique, Université Paris Sciences et Lettres, Université Paris-Saclay, Université Paris-Sud, Seoul National University Children's Hospital, Centre de Recherche en Cancérologie de Lyon, Hospices Civils de Lyon, Université Claude Bernard Lyon 1, Children's Memorial Health Institute, Universidade de São Paulo, Hospital Sant Joan de Déu Barcelona, Johns Hopkins University, Johns Hopkins Medicine, Emory University, Kitasato University, Erasmus MC, McMaster University, Neurological Surgery, University of Pittsburgh, Mayo Clinic in Arizona, Fred Hutch Cancer Center, University of Debrecen, Masaryk University, Vanderbilt University Medical Center, Washington University in St. Louis, University of California, Los Angeles, Semmelweis University, Chinese University of Hong Kong, University of Alabama at Birmingham, Chonnam National University Hwasun Hospital, Duke University, NYU Langone Health, University of Colorado Denver, Asan Medical Center, University of Ulsan, Istituto Giannina Gaslini, Universität Hamburg, University Medical Center Hamburg-Eppendorf, Osaka National Hospital, Centro Hospitalar Lisboa Norte, Hospital de Santa Maria, Centro Medico Nacional Siglo XXI, University Health Network, Toronto Western Hospital
Total RNA samples (2 µg) were arrayed into 96-well plates
DTTP was replaced with dUTP in the dNTP mix
dependent variables
Polyadenylated mRNA was purified
First-strand cDNA was synthesized
Second-strand cDNA was synthesized
The cDNA was quantified and checked for quality before fragmentation
Plate-based libraries were created
The libraries were sequenced using Illumina HiSeq 2000 or 2500, 2 × 100 PE lanes, with v3 chemistry and HiSeq Control Software version 2.0.10
control variables
Total RNA samples (2 µg) were arrayed into 96-well plates
Polyadenylated mRNA was purified with a MultiMACS mRNA isolation kit as per the manufacturer's instructions
First-strand cDNA was synthesized using a SuperScript cDNA Synthesis kit with random hexamer primers
The SuperScript cDNA Synthesis protocol was used for second-strand cDNA synthesis
The second strand was digested with UNG (Uracil-N-Glycosylase, Life Technologies, USA) in the post-adapter ligation reaction
Annotations
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