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Sample GSM7903231

Query DataSets for GSM7903231

Status

Public on Feb 26, 2024

Title

WT apex bio bio rep 2

Sample type

SRA

Source name

apex

Organism

Medicago truncatula

Characteristics

tissue: apex
developmental stage: 15 days old
genotype: R108
genetic modification: wild type

Treatment protocol

Fully expanded trifoliate leaves (leaf) or shoot apices (apex) were harvested from 15 day old Mtsoc1a and WT. Two trifoliate leaves, one leaf taken from each of two plants, were pooled to make up one biological replicate; with three biological replicates harvested for each genotype. For the RNA-seq on the Mtsoc1-4 mutant, the upper portion of three primary stems with their shoot apex, from three plants, were pooled to make up one biological replicate, with three biological replicates harvested for each genotype.

Growth protocol

Medicago seeds were first scarified in between two pieces of sandpaper, sterilized in chlorine solution for 5-10 minutes and germinated overnight at 15°C in the dark. Germinated seeds were placed on moist filter paper and stored at 4°C in the dark for 3 weeks. Germinated seeds with or without vernalisation were planted in seed raising mix (Daltons Limited, NZ) and placed on sub-irrigated rockwool mats. Seedlings were transplanted after 11-14 days to 2L pots of soil mix consisting of potting mix, vermiculite and number 2 sand. Plants were grown in controlled rooms in long day (LD, 16 hours light/8 hours dark) or short day (SD, 8 hours light/16 hours dark) photoperiods at 22°C.

Extracted molecule

total RNA

Extraction protocol

Fully expanded trifoliate leaves (leaf) or shoot apices (apex) (15-100 mg), were harvested from plants grown under VLD conditions at Zeitgeber time 4 (4 h after dawn). Tissues were snap frozen with metal beads and homogenised using a Geno/Grinder (New Jersey, USA) into powder in liquid nitrogen. Total RNA was extracted using the RNeasy Plant Mini Kit (Qiagen, Germany) following the manufacturer’s protocol. The quantity and quality of RNA were checked by Bioanalyzer 2100 (Agilent Technologies, USA).
The RNA of Mtsoc1a mutant and wild type R108 were constructed into 12 strand-specific mRNA libraries with polyA enrichment and were subjected to Illuminia NovaSeq 6000 paired-end 150 bp sequencing by Novogene. The RNA of triple Mtsoc1-4 mutant were constructed into three strand-specific mRNA libraries with polyA enrichment and sequenced on Illumina platform NovaSeq 6000, paired-end 150 bp by Novogene (Hong Kong).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

Data processing

The quality of the sequenced raw reads (FASTQ files) was assessed and the trimming of the reads was done using Fastp (version 0.21) (Chen et al., 2018). Reads were trimmed from the 3′ end where quality dropped below a PHRED score of 20 and any remaining reads under 36 bp in length were also excluded.
The trimmed reads were mapped to the Mt4.0v2 transcriptome (Young et al., 2011; Tang et al., 2014) using Salmon (v0.8.2) (Patro et al., 2017). The resulting count tables, in which abundance is measured as Transcripts per Million (TPM), were then imported into R (R Core Team, 2018) using the tximport package (v1.12.0) (Soneson et al., 2015). Normalisation and differential expression analysis visualization were performed using DESeq2 (v1.24.0) (Love et al., 2014). Differentially expressed transcripts were filtered-out using a cut-off of adjusted p-value ≤ 0.05 and log2fold-change ≥ 1 or ≤-1.
Assembly: MedtrA17_4.0 (Mt4.0v2_GenesCDSSeq_20140818_1100.fasta)
Supplementary files format and content: Excel file including raw counts for each sample
Supplementary files format and content: excel file including normalised counts for each sample

Submission date

Nov 15, 2023

Last update date

Feb 26, 2024

Contact name

Joanna Putterill

E-mail(s)

j.putterill@auckland.ac.nz

Organization name

The University of Auckland

Department

School of Biological Sciences