Similar protocols

Pipeline publication

[…] tion plants three weeks after germination., Fresh-frozen cotyledons and first true-leaves (before the second true leaf emergence) of B. villosa and B. oleracea were collected for RNA sequencing from seedlings (5–7 plants per replicate) for 3 biological replicates. RNA was extracted using standard methods, then developed into libraries using a TruSeq RNA sample preparation kit (Illumina, San Diego, CA, USA). Library sequencing (100 cycles) was conducted from both ends on an Illumina HiSeq 2000. A total of 51 Gb of B.oleracea reads (Cotyledons; 26 Gb and true leaves; 25 Gb) and 60 Gb of B.villosa reads (Cotyledons; 33 Gb and true leaves; 27 Gb) were obtained. The RNAseq data was trimmed using trimmomatic ver.0.30 with minimum quality score of 15, removing the first 12 bp, and a minimum length of 20, which was then aligned to the genome of B. oleracea with TopHat ver. 2.0.7 (with parameters, including minimum intron length (i) of 20, maximum intron length (I) of 11000, and distance between pair ends (r) of 30) . The aligned RNAseq reads were assembled into transcripts and their relative abundance was estimated as fragments per kilobase of exon per million fragments mapped (FPKM) using Cufflinks software . Differential expression and statistical analysis of individual gene copies was conducted using Cummerbund package in R; Raw data from the RNAseq experiment was deposited to NCBI with SRA accession number, SRP035213., First true leaves were collected separately from three individual plants at the same stage as mentioned above for B. villosa for each of the four other species mentioned under plant material. Total RNA was extracted from the seedling leaves (three independent preparations per species) with a commercial RNA-Easy mini kit (Qiagen, Valencia, CA, USA) and cDNA synthesized using Superscript II™ (Invitrogen, Carlsbad, CA) according to manufacturer’s instructions. Quantitative real time-PCR (Q-PCR) was conducted using a Platinum SYBR Green Super Mix-UD […]

Pipeline specifications

Software tools Trimmomatic, TopHat, Cufflinks, CummeRbund
Organisms Arabidopsis thaliana, Brassica oleracea