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Sample GSM5694743

Query DataSets for GSM5694743

Status

Public on Nov 20, 2021

Title

Blue needles Indv 2

Sample type

SRA

Source name

Samples from adult trees with blue needles

Organism

Abies pinsapo

Characteristics

tissue: Needles
geo_loc_name: Spain: Sierra de las Nieves
phenotype: blue

Treatment protocol

The samples were harvested from adult trees. The trees were selected depending on the color of their needles, blue or green. Developing needles were avoided because their pigment composition.

Growth protocol

Field-grown adult trees from Sierra de las Nieves (Yunquera, Spain).

Extracted molecule

total RNA

Extraction protocol

Total root RNA from maritime pine seedlings was isolated following the protocol described by Liao et al. (2004, Preparative Biochemistry & Biotechnology 34: 209–214) and modified by Canales et al. (2012, Frontiers in Plant Science 3: 100). The RNA concentration and purity were determined via spectrophotometry on a Nanodrop ND-1000 (Thermo Scientific, Waltham, MA, USA). Purity was determined through the 260/280 and 260/230 ratios. RNA quality was also determined in a Bioanalyzer 2100 (Agilent, Santa Clara, CA, USA). The concentration was verified with a Qubit 4 Fluorometer (Invitrogen, Paisley, UK) and Qubit RNA BR, Broad-Range, Assay Kit (Invitrogen, Paisley, UK). Samples with a RIN value > 7 were selected to mRNA isolation. The polyA-RNA isolation was performed using Dynabeads™ mRNA Purification Kit (Invitrogen, Paisley, UK) following the manufacturer’s instructions. This process was carried out twice per sample to avoid rRNA contamination. polyA-RNA quality was determined in a Bioanalyzer 2100 (Agilent, Santa Clara, CA, USA). The concentration was verified with a Qubit 4 Fluorometer (Invitrogen, Paisley, UK) and Qubit RNA HS, High Sensitivity, Assay Kit (Invitrogen, Paisley, UK).
RNA-seq was carried out by Novogen (UK). mRNA was enriched using oligo(dT) beads. First, the mRNA was fragmented randomly by adding fragmentation buffer, then the cDNA was synthesized by using mRNA template and random hexamers primer, after which a custom second-strand synthesis buffer (Illumina) , dNTPs, RNase H and DNA polymerase I were added to initiate the second-strand synthesis. Second, after a series of terminal repair, A ligation and sequencing adaptor ligation, the double-stranded cDNA library was completed through size selection and PCR enrichment. 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 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

Description

Blue2

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: Assembled reference transcriptome
Supplementary_files_format_and_content: Tab-delimited text files include CPM values for each sample.The column ID is the sequence identifier.

Submission date

Nov 18, 2021

Last update date

Nov 20, 2021

Contact name

Rafael A Cañas

E-mail(s)

rcanasp@gmail.com

Organization name

Universidad de Málaga

Department

Biología Molecular y Bioquímica