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Sample GSM8488584

Query DataSets for GSM8488584

Status

Public on Sep 05, 2024

Title

Bd21, root, nitrate, rep2

Sample type

SRA

Source name

Whole root

Organism

Brachypodium distachyon

Characteristics

tissue: Whole root
genotype: Bd21
treatment: nitrate nutrition

Growth protocol

Each sample represents a pool of 12 seedlings of B. distachyon Bd21 grown in a hydroponic tank of 4.5 L with ammonium or nitrate as source of nitrogen. Plants were grown during 19 days with 2.5 mM N supplied as 1.25 mM (NH4)2SO4 or 1.25 mM Ca(NO3)2 for ammonium and nitrate nutrition, respectively.

Extracted molecule

polyA RNA

Extraction protocol

Total RNA was extracted using the Nucleospin RNA plant kit (Macherey-Nagel)
One 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 (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, adaptorligated 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.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2000

Description

Bd21_N2

Data processing

Paired-end clean reads were mapped to the reference genome using HISAT2 software. Htseq was used to count the read numbers mapped to each gene.
Assembly: v3.1
Supplementary files format and content: comma-separated values file includes raw counts for each sample

Submission date

Aug 29, 2024

Last update date

Sep 05, 2024

Contact name

Daniel Marino

E-mail(s)

[email protected]

Organization name

University of the Basque Country (UPV/EHU)

Street address

Barrio Sarriena s/n