title: 29BPG16A
organism: Fragaria x ananassa
description: Protocols: Fruits were harvested at the stage of full ripeness, frozen in liquid nitrogen and stored a -30ºC until used. Transgenic plants were grown in a confined greenhouse under natural temperature and photoperiod Total RNA was isolated from independent pools of ripe fruits in accordance with Gambino et al. (Phytochem. Anal. 19, 520-525, 2008). Achenes were removed from ripe fruit and the receptacle RNA was extracted and puriﬁed RNA-seq library and sequencing was performed by Novogene A total amount of 1 μg RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer's recommendations and index codes were added to attribute sequences to each sample. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer (5X). First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (RNase H-). Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3' ends of DNA fragments, NEBNext Adaptor with hairpin loop structure were ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 150~200 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). Then 3 μl USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37 °C for 15 min followed by 5 min at 95 °C before PCR. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. At last, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system.
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2021-01-21T04:12:56Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2020-01-27T12:10:13Z
attributes.tag: External Id
attributes.value: SAMEA6503702
attributes.tag: INSDC center name
attributes.value: Dept. Botanica y Fisiologia Vegetal Universidad de Malaga
attributes.tag: INSDC first public
attributes.value: 2021-01-21T04:12:56Z
attributes.tag: INSDC last update
attributes.value: 2020-01-27T12:10:13Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-8736:29BPG16A
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: common name
attributes.value: strawberry
attributes.tag: cultivar
attributes.value: Chandler
attributes.tag: developmental stage
attributes.value: fruit ripe stage
attributes.tag: genotype
attributes.value: 29BPG16
attributes.tag: individual
attributes.value: transgenic line no 16
attributes.tag: organism part
attributes.value: fruit
attributes.tag: phenotype
attributes.value: FaPG1 and FaPG2 downregulated
attributes.tag: sample name
attributes.value: E-MTAB-8736:29BPG16A
attributes.tag: scientific_name
attributes.value: Fragaria x ananassa