title: Lolium perenne, ecotype Falster
organism: Lolium perenne
description: Protocols: 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). 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. 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.
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2013-04-18
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2013-03-21
attributes.tag: External Id
attributes.value: SAMEA1877883
attributes.tag: INSDC center alias
attributes.value: Crop Genetics and Biotechnology, Dept. of Molecular Biology and Genetics, Aarhus University, Denmark
attributes.tag: INSDC center name
attributes.value: Crop Genetics and Biotechnology, Dept. of Molecular Biology and Genetics, Aarhus University, Denmark
attributes.tag: INSDC first public
attributes.value: 2013-04-18T17:03:35Z
attributes.tag: INSDC last update
attributes.value: 2013-03-21T09:56:41Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-1556:Lolium perenne, ecotype Falster
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: organism part
attributes.value: leaf
attributes.tag: sample name
attributes.value: E-MTAB-1556:Lolium perenne, ecotype Falster
attributes.tag: specimen with known storage state
attributes.value: frozen specimen
attributes.tag: strain
attributes.value: Falster