title: Sample_6
organism: Phoenix dactylifera
description: Protocols: For our transcriptomic sequencing of date palm (Phoenix dactylifera), young leaf and root tissues were collected from both healthy and stressed plants in the greenhouse using sterilized scissors. The samples were immediately flash-frozen in liquid nitrogen and stored at -80°C until RNA extraction. To ensure data accuracy, sterility was maintained throughout the collection process, and metadata, including plant condition, treatment, and timepoint, was recorded. Date palm plants samples were grown in a controlled greenhouse environment under natural photoperiod conditions, with temperatures maintained between 25-30°C during the day and 18-22°C at night. Healthy plants were irrigated with sterile water, while stressed plants were treated with 300 mM NaCl after 72 hours, ensuring adequate soil moisture in all samples This RNA extraction protocol employs CTAB reagent and ethanol precipitation for purifying total RNA from plant tissue. The process begins with preparing a lysis buffer by mixing 2% CTAB reagent with beta-mercaptoethanol, followed by grinding approximately 50 mg of plant tissue using liquid nitrogen and suspending it in the CTAB reagent. The lysate is incubated at 65°C for 15 minutes to facilitate nucleoprotein dissociation before centrifugation to remove debris. The supernatant is then subjected to chloroform extraction and centrifuged to separate the aqueous phase containing RNA. Further purification is achieved through phenol-chloroform extraction, followed by an additional chloroform wash. RNA is precipitated using isopropanol and incubated at -20°C for two hours before centrifugation to pellet the RNA. The pellet is then washed twice with 75% ethanol, air-dried, and finally dissolved in DEPC-treated water to obtain purified RNA for downstream applications. The strand-specific transcriptome library construction protocol is designed to obtain transcript sequences and determine transcript orientation, providing insights into gene structure and regulation. The process begins with mRNA isolation using oligo(dT)-attached magnetic beads to purify mRNA from total RNA. The mRNA is then fragmented into smaller pieces using fragmentation reagents. First-strand cDNA synthesis is performed using primers and thermal cycling, followed by second-strand cDNA synthesis, where dUTP is incorporated instead of dTTP to facilitate strand specificity. The resulting double-stranded cDNA undergoes end repair and A-tailing to ensure proper ligation of sequencing adapters. Adaptor ligation is then carried out enzymatically, and the ligated products are purified using magnetic beads. The second-strand template is digested with UDG enzyme before PCR amplification to enrich the library. The amplified library is validated using an Agilent 2100 bioanalyzer. Following quality control, circularization of double-stranded PCR products is performed to generate single-stranded circular DNA (ssCir DNA), forming the final library. The library is then amplified using phi29 polymerase to create DNA nanoballs (DNBs), which are loaded onto a patterned nanoarray for sequencing using combinatorial Probe-Anchor Synthesis (cPAS) technology, generating single-end 50 bp or paired-end 100/150 bp reads.
attributes.tag: ENA first public
attributes.value: 2026-03-20
attributes.tag: INSDC center name
attributes.value: UAEU
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-16561:Sample_6
attributes.tag: age
attributes.value: 6
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: collection date
attributes.value: not collected
attributes.tag: common name
attributes.value: date palm
attributes.tag: cultivar
attributes.value: Khanizi
attributes.tag: developmental stage
attributes.value: seedling development stage
attributes.tag: disease
attributes.value: normal
attributes.tag: genotype
attributes.value: Khnaizi (Phoenix dactylifera cultivar)
attributes.tag: geographic location (country and/or sea)
attributes.value: not collected
attributes.tag: isolate
attributes.value: not applicable
attributes.tag: organism part
attributes.value: root
attributes.tag: read type
attributes.value: Forward
attributes.tag: sample name
attributes.value: E-MTAB-16561:Sample_6
attributes.tag: scientific_name
attributes.value: Phoenix dactylifera