| Title |
Root nodule library |
| Library name |
MtBB |
| Library type |
EST, 5'-3' unidirectional |
| Organism |
Medicago truncatula |
| Tissue type |
Root nodule |
| Developmental stage |
Plants were grown in an aeroponic chamber on nitrogen-rich medium for 21 days. Three days before inoculation with Sinorhizobium meliloti, the medium was replaced by N-free medium. Root nodules (+ short adjacent root segments) were harvested 4 days post inoculation. |
| Vector |
pBluescript SK- |
| Lab. host |
E. coli SOLR |
| Cloning site R1 (5') |
EcoRI |
| Cloning site R2 (3') |
XhoI |
| 5' Adaptor sequence |
5'-AATTCGGCACGAGG-3' |
| 3' Adaptor sequence |
5'-GAGAGAGAGAGAGAGAGAACTAGTCTCGAGT(17)-3' |
| Description |
For each library, cDNA was prepared from polyA+ enriched RNA. The cDNA was directionally ligated into Uni-Zap XR vector from Stratagene (site1: EcoRI; site2: XhoI) and packaged using Gigapack Gold packaging extracts. Plasmids (pBluescript SK) containing cDNA inserts were mass-excised from phage stocks using ExAssist helper phage and propagated in SOLR cells. |
| 5' Sequencing primer |
TM |
| 5' Sequencing primer sequence |
5'-ATTAACCCTCACTAAAGGGA-3' (equivalent to T3 primer) |
| 3' Sequencing primer |
CM |
| 3' Sequencing primer sequence |
5'-TAATACGACTCACTATAGGG-3' (equivalent of T7 primer) |
| Other sequencing primer |
|
| Other sequencing primer sequence |
|
| Potential source of contaminant |
|
| Additional comments |
Clone ordering and sequencing was performed by the Centre national de sequencage (Genoscope, Evry, France). Due to the directional cloning of cDNA inserts into the Uni-Zap XR vector, EST sequences obtained with the forward primer (from T3 side of the cloning site) corespond to cDNA 5' ends, and those obtained with the reverse primer (from T7 side of the cloning site; sequence then converted into reverse complement) correspond to cDNA 3' ends. However, it should be born in mind that we detected the presence of a few % inserts cloned in the reverse orientation. |
| Library developer |
Pascal Gamas, Etienne-Pascal Journet |
| Publication reference |
http://sequence.toulouse.inra.fr/Mt/public/cDNA_library_construction.html |