E-MTAB-2623:Falster_leaf_9weeksVERN
Lolium perenne
Protocols: Plants from Lolium perenne genotypes Falster and Veyo 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 (15 degrees C and 8 hours day length). A vernalization treatment was carried out for 9 weeks, at a temperature of 6 degrees C and 8 hours day length. Leaf material was collected before the start of vernalization, after 2 days, 4 weeks, and 9 weeks of vernalization. Enriched meristem samples were collected after 9 weeks of vernalization. This was followed by a secondary induction treatment, carried out at a temperature of 20 degrees C and 16 hours day length. Leaf samples were collected after seven days of long day conditions, while enriched meristem samples was collected after one and seven long days. Leaf and/or enriched meristem material was collected from 4 individual plants in case of each genotype, and each condition. Equal amount of plant material was pooled together in one biological sample, from which one RNA extraction was performed. 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). 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.
ENA checklist
ERC000011
INSDC center alias
Crop Genetics and Biotechnology, Dept. of Molecular Biology and Genetics, Aarhus University, Denmark
INSDC center name
Crop Genetics and Biotechnology, Dept. of Molecular Biology and Genetics, Aarhus University, Denmark
INSDC first public
2014-09-17T17:04:08Z
INSDC last update
2014-06-05T14:20:05Z
INSDC status
public
SRA accession
ERS474016
Sample Name
ERS474016
alias
E-MTAB-2623:Falster_leaf_9weeksVERN
broker name
ArrayExpress
developmental stage
vegetative phase
genotype
Falster
organism
Lolium perenne
organism part
leaf
secondary description
The upper two thirds of the most recently developed and fully expanded leaf was harvested at each time point
specimen with known storage state
frozen specimen
title
Falster_leaf_9weeksVERN