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Sample GSM2070595

Query DataSets for GSM2070595

Status

Public on Jun 30, 2017

Title

Hypocotyl_Pith

Sample type

SRA

Source name

Hypocotyl Pith

Organism

Pinus pinaster

Characteristics

tissue: Hypocotyl Pith
provenance: Oria
developmental stage: 1 month-old seedling
culture: In soil
culture place: Culture chamber at Universidad de Malaga (Malaga, Spain)

Treatment protocol

The seedlings were cut and tissue sections of 5 mm were mounted in a specimen holder with optimal cutting temperature (OCT) embedding medium (Tissue-Tek, USA) and snap-frozen in liquid nitrogen for cryostat sectioning. The frozen samples were stored at -80ºC until use.

Growth protocol

Maritime pine seeds (Pinus pinaster Ait.) from the Oria provenance were provided by Centro de Recursos Genéticos Forestales “El Serranillo” (Ministerio de Medio Ambiente y Medio Rural y Marino, Spain). 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 cultivated in a growth room at 25˚C and 50/70% relative humidity with a 16/8-h photoperiod and watered twice a week with distilled water. Fourteen days after imbibitions, seedlings were individually transferred to a 0.2-L pot with soil and watered twice a week with distilled water. The seedlings were harvested 1 month after the emergence of the shoots over the vermiculite.

Extracted molecule

total RNA

Extraction protocol

The Laser Capture Microdissection (LCM) procedure was carried out as previously described in Cañas et al. (Tree Physiol. 34:1278-1288). The microdissection samples were placed into the caps of 0.5 mL tubes containing 10 L lysis buffer from the RNAqueous-Micro RNA Isolation Kit (Ambion, USA). Before RNA extraction all the samples were placed at -80ºC. All the RNA extractions from the microdissection of the samples were made using the standard-volume protocol (non-LCM) for the RNAqueous-Micro RNA Isolation Kit (Ambion, USA). RNA quality and first quantification was assessed using the RNA Pico Assay for the 2100 Bioanalyzer (Agilent, USA). The RNA samples with a RNA integrity number (RIN) higher than 7 were used for the subsequent mRNA amplification and 454 pyrosequencing.
As the amounts of RNA isolated form LCM samples were not enough for 454 pyrosequencing, a previous cDNA synthesis and amplification was made using the Conifer RNA Amplification (CRA+) protocol described in Cañas et al. (Tree Physiol. 34:1278-1288). Transcriptome sequencing was performed at the Universidad de Málaga ultrasequencing facility using the GS-FLX+ platform with a GS FLX Titanium kit, Roche Applied Sciences (Indianapolis, IN, USA). Each sample was run in one half of a 454 PicoTiterPlate following the manufacturer’s sequencing protocol. The quantity of the cDNA libraries was determined using the Quant-iT™ PicoGreen® dsDNA Kit (Invitrogen, Paisley, UK). The quality of the cDNA libraries was determined using the Agilent High Sensitivity DNA Kit in the 2100 Bioanalyzer (Agilent, CA, USA). The runs were analysed using the Roche GS-FLX+ software.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

454 GS FLX Titanium

Description

Gene expression in Hypocotyl Pith
HP

Data processing

We assembled the transcriptome for P. pinaster from the 454 sequencing reads by using Newbler (v2.8.1). Before feeding reads to Newbler, we removed reads with adapter sequences and reads shorter than 75bp through seqclean. Newbler then assembled the remaining rest reads in six rounds for P. pinaster, until all of the over-represented sequences were removed. CD-HIT-EST (Weizhong Li & Godzik 2006) then clustered the Newbler assemblies in each isogroup, which stands for unique transcriptional locus in Newbler assembly result, and selected the longest transcript, which had to be ≥ 150 bp, to represent each isogroup.
For the read mapping the raw data was first checked for quality using the fastqc software.
Based on examination of the output quality plots it was decided to clip off the first 35bp of each read. To that end, we used the FastX toolkit (fastx_trimmer).
Next the reads where filtered for overall quality using fastq_quality_filter with a minimum Q-score of 30 and minimum remaining length of 60%.
The remaining reads where subsequently used for read mapping with the BWA software (default parameter settings) against the reference transcriptome composed by 206,574 contigs.
Contigs with at least 25 reads in a sample were conserved, the rest of contigs were filterd. Filtering and normalization were done using edgeR package for R.
Gene co-expression analyses were made using the WGCNA package for R.
Genome_build: Reference transcriptome composed by 206,574 contigs from PROCOGEN and SUSTAINPINE pojects and Genbank (http://www.bmbq.uma.es/fmp/Datos/ALL_Procogen.fasta)
Supplementary_files_format_and_content: Tab-delimited text files include CPM values for each sample (AM, EN, YNM, YNV, CM, CV, HC, HV, HP, RC, RV, DRC, DRV and RM).The column ID_REF is the sequence identifier.

Submission date

Feb 24, 2016

Last update date

May 15, 2019

Contact name

Rafael A Cañas

E-mail(s)

rcanasp@gmail.com

Organization name

Universidad de Málaga

Department

Biología Molecular y Bioquímica