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Sample GSM1053998

Query DataSets for GSM1053998

Status

Public on Jun 05, 2013

Title

Control: Seed from ColA9 X Col-0 rep2

Sample type

SRA

Source name

Arabidopsis seeds

Organism

Arabidopsis thaliana

Characteristics

ecotype: ColA9 X Col-0
tissue: whole seeds
time: 3 days after pollination

Treatment protocol

Seeds were harvested fresh from ColA9 X A. arenosa, C24A9 X A. arenosa, ColA9 x Col-0 and C24A9 x C24 and frozen on dry ice. Total RNA was obtained using Plant RNA Reagent (Invitrogen, Carlsbad, California). Frozen tissue was placed in a 1mL Dounce Tissue Grinder (Omni International, Kennesaw, GA) with 200 μL Plant RNA Reagent. The tissue was ground for approximately 30 seconds and then the extract was transferred to a clean tube. The grinder was washed with 300 μL of Plant RNA Reagent, which was combined with the tissue extract. RNA extraction proceeded with the manufacturer’s protocol and followed by a DNAse I (Invitrogen) digest at room temperature for 60 minutes according to manufacturer’s recommendation to remove any DNA contamination. RNA was eluted in 20 μL DNAse-free water and concentration was determined using a NanoDrop 1000 Spectophotometer. RNA was stored at -80°C for downstream applications.

Growth protocol

All plants were grown under long day conditions (16hrs light at 21°C and 8hrs dark at 18°C) in a controlled environment facility at Davis, CA. For expression analysis, the female Col-0 (seed stock ID CS6673) and C24 (seed stock ID CS22620) diploid ecotypes of A. thaliana were hemizygous for a male sterility construct (Paul et al., 1992) and were gifts from Dr. Rod Scott (University of Bath, United Kingdom). These male sterile lines were selected to reduce contamination. Diploid Arabidopsis arenosa var. Strecno was given to the lab by Dr. M. Lysak. The male-sterile A. thaliana lines were used as seed parent and A. arenosa was used as the pollen donor for all interspecific crosses. One A. arenosa plant was used for all crosses in these experiments to reduce genetic variation due to heterozygosity in A. arenosa lines. Crosses were performed by pollinating fully developed stigmas by hand at 10am to reduce circadian biases; no emasculation was necessary because of the male sterility construct.

Extracted molecule

total RNA

Extraction protocol

Approximately 10μg of total RNA was processed in preparation for Illumina Sequencing using a homemade kit. All enzymatic reactions followed manufacture’s protocol unless otherwise noted. Messenger RNA was purified from total RNA using Dynabeads (Invitrogen, Carlsbad, California), which purify poly-A containing transcripts. Then, cDNA was synthesized with random priming using SuperscriptIII (Invitrogen) followed by heat inactivation for 5 minutes at 85°C. Second strand cDNA was then synthesized as follows: We combined 5x second strand buffer [200 mM Tris-HCl, pH 7; 22 mM MgCl2; 425 mM KCl], water, and 10mM dNTPs with the first strand mix. The reaction was incubated on ice for 5 minutes and then DNA polymerase I (NEB, Ipswich, Massachusetts) and RNAseH (NEB, Ipswich, Massachusetts) were added and the reaction was incubated at 16°C for 2.5 hours. The double stranded cDNA was cleaned using Agencourt AMPure XP (Beckman Coulter Genomics, Danvers, Massachusetts) with a 1.8:1 (v/v), AMPure to library ratio. After cDNA synthesis, each library was chemically fragmented using 1 μl of NEB’s dsDNA Fragmentase for 30 minutes at 37°C and cleaned with AMPure. Fragmentation was followed by end repair with NEB’s End Repair Module Enzyme Mix and A-base overhangs, which are needed for adaptor ligation, were added with Klenow (NEB). End repair and A-base addition were both followed by AMPure cleanup. Barcoded Illumina adaptors for controls and regular Illumina adaptors for controls were ligated at room temperature using NEB Quick Ligase. To remove adapter contamination in library sequencing, we size selected our libraries using AMPure. Changing the amount of AMPure buffer changes bead size selection. We used 0.1:1 (v/v), AMPure: library to obtain fragments (library + adaptor) greater than 300 bp and eluted in 20 μl Qiagen Elution Buffer . 10 μl of each library was enriched using 15 μl Phusion 2x HF master mix (NEB), 3 μl water, and 2 μl 5 μM premixed PE primers with the following reaction conditions: 30 sec at 98°C; 14 cycles of 10 sec at 98°C, 30 sec at 65°C, and 30 sec at 72°C; final extension with 5 min at 72°C. Enriched libraries were purified with AMPure (0.8:1 v/v; AMPure: library) and quality and quantity was assessed using Agilent BioAnalyzer. Libraries were sequenced according to manufacture’s instructions. The hybrids were sequenced using Illumina’s HiSeq 2000 (100-bp paired-end reads) and the controls were sequenced using Illumina’s HiSeq 2000 (Illumina, San Diego) (100-bp paired-end reads) or Illumina’s Genome Analyzer IIx (40-bp single-end reads).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2000

Description

Control: Seed from ColA9 X Col-0

Data processing

All sequence preprocessing was done with custom python scripts available at the Comai lab wiki (http://comailab.genomecenter.ucdavis.edu/index.php/Barcoded_data_preparation_tools). We chose to process forward reads only so as not to inflate count scores. Sequences were trimmed using 4 separate scripts to remove adaptors, remove barcodes, convert Illumina 1.5+ fastq to sanger, and remove ambiguous bases. Reads that were shorter than 34-35 bp were discarded along with base quality score lower than Phred 20.
To assess gene expression, each biological replicate was aligned separately to both TAIR10 cDNA (available for download at ftp://ftp.arabidopsis.org//home/tair/Sequences//blast_datasets/TAIR10_blastsets/TAIR10_cdna_20101214) using Burrows-Wheeler Aligner (BWA) version 0.5.8c (Li and Durbin, 2009).
After alignment, read counts were generated using a custom python script.Sequences that mapped to more than one gene model of the same gene were saved and counted as one read count while sequences that mapped to more than one gene were excluded.
Genome_build: TAIR10 cDNA (all gene models)
Supplementary_files_format_and_content: .txt (read count per locus file generated from the .sam files)

Submission date

Dec 17, 2012

Last update date

May 15, 2019

Contact name

Luca Comai

E-mail(s)

lcomai@ucdavis.edu

Phone

(530) 752-8485

Organization name

UC Davis