normalization data transformation protocol
The option for no biological replicates from DESeq version 1.4.1 was used to identify differentially expressed sequences in a pairwise comparison between the time points.
high throughpt sequence alignment protocol
In order to assess the expression level of each transcript at the different time points, we used RSEM version 1.1.11 to generate the read count data, by mapping the RNA-Seq reads onto the assembled transcripts. Up to one mismatch was allowed in the seed region (the first 25 bp) of the reads in the mapping process (--bowtie-n 1).
high throughpt sequence alignment protocol
Single end reads with a length of 50 bp were generated in the RNA-Seq sequencing. The quality of the resulted reads was assessed using FastQC. Having a Pearson’s correlation coefficient value above 0.99, the three technical replicates were pooled before further analysis, for a higher sequencing depth. A de novo transcriptome assembly was constructed using Trinity software (release Aug.2011) and only assembled transcripts with a minimum length of 100 bp were selected for further analysis. Homology based ORF prediction was done with OrfPredictor using BLASTX results against annotated proteins from rice, B. distachyon, maize, and sorghum (e-value<1e-5) as a guide. Redundancy in the data sets were subsequently removed using CD-HIT with the parameters -c 0.99 –n 5, i.e. a 99% sequence identity threshold and a word size of 5. Finally, CDS sequence sets were filtered using functions in the SeqinR package in R to only contain proteins longer than 30 amino acids (aa) with a start codon, allowing for no undetermined aa-residues caused by N’s in the transcript sequences.
nucleic acid sequencing protocol
Equimolar amounts of each of the libraries were pooled, diluted in buffer EB (QIAGEN) to 10 nM, denaturated with 2 N NaOH to a final DNA concentration of 1.0 nM, diluted to 10 pM with pre-chilled Hybridization buffer (Illumina), and were subsequently loaded into the lanes of a flow cell (v4). A single lane of the Multiplexing PhiX Control library (Illumina) was included in the flow cell and used to estimate phasing and matrix correction for all other lanes, to compensate for the skewed base compositions in the lanes containing the mRNA-Seq libraries. Clustering was conducted on the Illumina Cluster Station followed by 76+7+76 cycle sequencing on the Illumina Genome Analyzer (version IIx, SCS 2.6.26/RTA 126.96.36.199), using the Genomic DNA Sequencing Primer, the Index Seq Primer, and the Multiplexing Rd2 Seq Primer, in combination with clustering and sequencing kits (SBS v4), all supplied by Illumina. RTA output was transferred to the pipeline computer (DELL PowerEdge 2900, 8 cores (2 x 4 CPU, 2.6 GHz, 16 GB RAM, 4.7 TB hard drive) during the run, and analyzed using the CASAVA software (version 1.6, Illumina), generating pass filtering fastq files with Qphred+64 quality values. One biological replicate, representing plant material pooled from four individual plants, and three technical replicates were used in the Illumina Genome Analyzer II RNA-Seq process.
nucleic acid library construction protocol
10 ug of total RNA from each sample, representing material pooled from four individual plants, were used for the mRNA-Seq library construction, combining the protocol supplied with the mRNA-Seq Sample Prep Kit (Illumina, protocol version 1004898 Rev. D, September 2009) and the Multiplexing Sample Preparation Oliogonucleotide Kit. Poly-A containing mRNA was purified using poly-T oligonucleotide-attached magnetic beads and fragmented using divalent cations under elevated temperature. The RNA fragments were then copied into first strand cDNA using reverse transcriptase and random hexamer primers, followed by second strand cDNA synthesis using DNA Polymerase I and RNaseH. The short cDNA fragments were end-repaired using T4 DNA polymerase and Klenow DNA polymerase and a single A base were added to the cDNA fragments by using 3prime to 5prime exo-nuclease. This was followed by ligation of Illumina adaptors from the Multiplexing Sample Preparation Oliogonucleotide Kit and by gel purification of fragments of approximately 200 bp in size. The libraries were finally duplexed (index #6: GCCAAT or index #12: CTTGTA) and enriched by PCR for 18 cycles using the primers from the Multiplexing Sample Preparation Oliogonucleotide Kit. The index is a unique 6 bp sequence which is sequenced in a separate read, after sequencing the insert. The concentrations of the libraries were determined using a Qubit fluorometer (Invitrogen), and the size and purity of the libraries were determined with the Agilent 2100 Bioanalyzer in combination with the Agilent DNA 1000 Kit.
A vernalization treatment was carried out for 9 weeks, at a temperature of 6C and 8 hours day length. Leaf material was collected before the start of vernalization, after 2 days, 4 weeks, and 9 weeks of vernalization, between 9-10 am.
Plants from Lolium perenne ecotype Falster were grown in the greenhouse for 75 days after cloning, and were subsequently transferred to the climate chamber for 58 days under simulated fall conditions (15C and 8 hours day length).
nucleic acid extraction protocol
Total RNA was extracted using the RNeasy Plant Mini kit (Qiagen) following the manufacturers protocol. The RNA quality and concentration were assessed with the Agilent RNA 6000 Nano kit on the Agilent 2100 Bioanalyzer (Agilent Technologies).