BGISEQ-500 paired end sequencing; Differential gene expression in roots of tcp9 and tcp20 mutants of Arabidopsis thaliana upon Heterodera schachtii infection as measured by RNAseq
Differential gene expression in roots of tcp9 and tcp20 mutants of Arabidopsis thaliana upon Heterodera schachtii infection as measured by RNAseq
Root systems were separated from leaves and removed from the medium. The root systems were immediately snap frozen in liquid nitrogen. Before processing to nucleic acid extraction, root tissue was ground in liquid nitrogen. Plants (Col-0, tcp9, tcp20, and tcp9tcp20) were grown on modified Knop's medium for 4 days in 16/8 hours light/dark at 21 degrees Celsius. After 4 days plants were (mock) inoculated with Heterodera schachtii. Treatments ended 24, 48 or 72 hours after the start of the inoculation. Approximately 80 plants/sample were collected and the experiment was repeated three times. The Maxwell® 16 Tissue LEV plant RNA kit was used for mRNA isolation with a Maxwell® AS2000 instrument (Promega), following the manufacturer's protocol. After the total RNA extraction and DNase I treatment, magnetic beads with Oligo (dT) are used to isolate mRNA. Mixed with the fragmentation buffer, the mRNA is fragmented into short fragments. Then cDNA is synthesized using the mRNA fragments as templates. Short fragments are purified and resolved with EB buffer for end reparation and single nucleotide A (adenine) addition. After that, the short fragments are connected with adapters. The suitable fragments are size-selected for the PCR amplification as templates. During the QC steps, Agilent 2100 Bioanaylzer and ABI StepOnePlus Real-Time PCR System are used in quantification and qualification of the sample library.