Computational protocol: WRINKLED1, A Ubiquitous Regulator in Oil Accumulating Tissues from Arabidopsis Embryos to Oil Palm Mesocarp

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Protocol publication

[…] Developing seeds were collected from liquid nitrogen frozen siliques at 7-8, 9-10, and 11-12 d after flowering. Siliques were opened over dry ice and frozen seeds were separated from the silique walls by filtering through a liquid nitrogen cooled sieve into a tube on dry ice. Approximately 100 mg of developing seeds was finely ground and RNA extracted as described [] and analyzed for yield and quality by capillary electrophoresis (Agilent 2100). Libraries for sequencing were prepared from 2–4 µg total RNA using Illumina TruSeq RNA kits and sequenced with Illumina HiSeq2000. Reads (50 nt) were trimmed, filtered and aligned to TAIR10 using TopHat v 1.4.1 (Parameters: --no-novel-juncs; -G TAIR10.gff) and Bow tie v 0.12.7. Cufflinks v 2.0.2 was used to generate gene FPKM expression measures. The results were also loaded into a genome browser for inspection. Three different read mappings were performed to assess WRI1 alternative splice forms. Samples were aligned to TAIR10 using the CLC Genomics, Map Reads to Reference Tool and Large Gap Read Mapping Tool (Parameters: Mismatch cost 2; Insertion cost 3; Deletion cost 3; Similarity 0.9; Length fraction 0.9). 117 million RNASeq reads for roots (SRR331219,SRR331224) and 58.4 million for flowers (SRR388668, SRR388670, SRR013413, SRR013416) were downloaded from the NCBI short read archive and mapped to TAIR10 as described above. […]

Pipeline specifications

Software tools TopHat, Cufflinks
Databases TAIR
Application RNA-seq analysis
Organisms Arabidopsis thaliana