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Sample GSM5342173

Query DataSets for GSM5342173

Status

Public on May 28, 2021

Title

Root meristem at time 24 ammonium 3 mM experiment 4 [A4T24_3_RM]

Sample type

SRA

Source name

Root meristem at time 24 ammonium 3 mM

Organism

Pinus pinaster

Characteristics

provenance: Segura-Alcaraz
developmental stage: 1 month-old seedling
treatment: Ammonium 3 mM
time: 24 hours
tissue: Root meristem
experimental replicate: 3

Treatment protocol

One-month old maritime pine seedlings were used for this experiment. Pine seedlings were randomly subdivided into three different groups, relocated into forestall seedbeds and irrigated with 80 mL of a solution that contains macro- and micronutrients without any nitrogen source (1.16 mM KCl; 0.63 mM KH2PO4; 0.35 mM MgSO4·7H2O; 0.17 mM CaCl2·H2O; 80 μM EDTA-FeSO4; 25.9 μM H3BO3; 10.2 μM MnCl2·4H2O; 1.3 μM ZnSO4·7H2O; 0.7 μM CuSO4·5H2O; 0.1 μM Na2MoO4·2H2O). After three days of acclimation, the control group was irrigated with 80 mL of water (C), the second group was supplied with 80 mL of 3 mM NH4Cl. Root samples were collected 24 houres after the irrigation. Seedling’s root tips were cut and tissues of 5 - 6 mm were imbibed in a specimen holder with Tissue‐Tek optimal cutting temperature embedding medium (Sakura Finetek, Alphen aan den Rijn, The Netherlands) and immediately frozen in liquid nitrogen for cryostat sectioning. LCM procedure: 1 day before cryosectioning, root tip samples were tempered at -20°C. Thirty micrometers thick sections were cut using a cryostat (CM1950, Leica Biosysyems, Wetzlar, Germany) at -20°C and mounted on PET-membrane 1.4 µm steel frame (Leica Biosysyems, Wetzlar, Germany). The PET-membrane containing samples were stored at −80°C until use. Before microdissection, samples were fixed in cold 70% ethanol for 30 seconds, embedding medium was removed in DEPC-treated water for 2 minutes and refixed in cold 100% ethanol for 2 minutes. Afterwards, samples were air dried and used for microdissection. A laser microdissector (LMD 7000, Leica, Wetzlar, Germany) was used for microdissection. The samples obtained were placed into 0.5 mL tube caps containing 20 μL of lysis buffer from the RNAqueous-Micro RNA Isolation Kit (Ambion, USA) and all samples were placed at −80°C. Four different tissue areas were isolated by microdissection approximatively corresponding to the root cap (RC), meristem (RM), developing cortex (RDC) and developing vessels (RDV) areas.

Growth protocol

Seeds from maritime pine (Pinus pinaster Ait.) from “Sierra Segura y Alcaraz” (Albacete, Spain) were provided by from the Área de Recursos Genéticos Forestales of the Spanish Ministerio de Agricultura, Pesca y Alimentación. Seeds were imbibed in distilled water for 24 h under continuous aeration and germinated in a plastic tray with vermiculite as a substrate. Seedlings were grown in vermiculite in plant growth chambers under 16 h light photoperiod, a luminal intensity of 100 μmol m−2 s−1, a constant temperature of 25 °C and watered twice a week with distilled water.

Extracted molecule

total RNA

Extraction protocol

All the RNA extractions from the microdissection procedure were carried out using manufacturer’s instruction protocol (non‐LCM) for the RNAqueous‐Micro RNA Isolation Kit (Ambion, USA). RNA quality, DNA contamination and first quantification were performed via RNA Pico Assay for the 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA). Quantification was verified via a Qubit RNA BR (Broad-Range) Assay Kit (Invitrogen, Paisley, UK). RNA samples with RNA integrity number (RIN) higher than 7 were used for subsequent RNA sequencing, mRNA amplification and cDNA synthesis.
The low input RNA-seq was carried out by Novogen (HK). The cDNA synthesis and amplification, and the library preparation was made with the SMART-Seq™ v4 Ultra™ Low Input RNA Kit for Sequencing (Takara, Mountain View, CA, USA) following the manufacturer’s instructions. RNA sequencing was made in a NovaSeq 6000 sequencer according to the manufacturer’s instructions for paired-end reads (Illumina, San Diego, CA, USA). The 24 samples were sequenced producing paired-end reads of 150 bp length.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

Data processing

Base calling was made with the NovaSeq 6000 RTA3 software (Illumina). Conversion of cbcl files to fastq files were made with bcl2fastq2 Conversion Software (Illumina)
The raw reads were trimmed (quality and contamination) using SeqTrimBB software (https://github.com/rafnunser/seqtrimbb). Only the pairs in which both reads passed the quality test were further analysed (Q>20).
The reads were assembled using Trinity 2.11.0 (Haas et al., 2013, Nature Protocols 8:1494-512). Contigs lower than 400 pb were eliminated, for the rest of contigs the redundancy was reduced using CD-HIT-EST software (Fu et al., 2012, Bioinformatics 28: 3150-3152).
The final transcriptome was used as the reference for the read mapping that was performed with BWA using the MEM option (Li and Durbin, 2009, Bioinformatics 25: 1754-1760). The read count was obtained with the phyton script sam2counts (https://github.com/vsbuffalo/sam2counts).
The transcript expression was normalised by cpm. Finally the transcripts were filtered; 2 cpm in at least 2 samples (Robinson et al., 2010, Bioinformatics 26: 139-140).
Genome_build: Assemble reference transcriptome
Supplementary_files_format_and_content: Tab-delimited text files include CPM normalized counts for each sample.The column id is the sequence/sample identifier.

Submission date

May 26, 2021

Last update date

May 28, 2021

Contact name

Rafael A Cañas

E-mail(s)

rcanasp@gmail.com

Organization name

Universidad de Málaga

Department

Biología Molecular y Bioquímica