organism: Arabidopsis thaliana
description: Protocols: Five-day-old wild type and abi4-1 seedlings vertically grown on continuous light were harvested for RNA extraction. Wild type and abi4-1 seeds were surfaced on MS medium. Seedlings were vertically grown for five days under continuous light at 85 umol s-1 m-2. Temperature in growth chamber was 22 to 24℃. RNA extraction assay was according to manual of Qiagen kit (74204). 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 UltraTM 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. 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 240 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: 2020-05-15T04:04:02Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2020-05-05T11:13:40Z
attributes.tag: External Id
attributes.value: SAMEA6813650
attributes.tag: INSDC center name
attributes.value: Photosynthesis Research Center, Key Laboratory of Photobiology, Institute of Botany, Chinese Academy of Sciences
attributes.tag: INSDC first public
attributes.value: 2020-05-15T04:04:02Z
attributes.tag: INSDC last update
attributes.value: 2020-05-05T11:13:40Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-9027:abi4-1
attributes.tag: age
attributes.value: 5
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: common name
attributes.value: thale cress
attributes.tag: developmental stage
attributes.value: cotyledon emergence stage
attributes.tag: ecotype
attributes.value: Col-0
attributes.tag: genotype
attributes.value: abi4-1 mutant
attributes.tag: organism part
attributes.value: whole organism
attributes.tag: sample name
attributes.value: E-MTAB-9027:abi4-1
attributes.tag: scientific_name
attributes.value: Arabidopsis thaliana