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Sample GSM2474320

Query DataSets for GSM2474320

Status

Public on Aug 21, 2017

Title

RNA-seq of Arabidopsis thaliana: Dark Grown [Dark-RNA3]

Sample type

SRA

Source name

RNA-seq_Dark Grown

Organism

Arabidopsis thaliana

Characteristics

ecotype background: Col0
cell type: 16h post-stratification cell culture
growth condition: Dark

Treatment protocol

Light grown cells were sampled 5 days or 16 hours after subculture. Dark grown cells were adapted to dark conditions for 2 weeks with a 7-day subculture. Dark grown cells were sampled 16-hour after the 2nd subculture. Cells were sampled by harvesting 30mL of cell culture and washed with autoclaved ultrapure water. Cells were frozen in liquid nitrogen and ground into a fine powder. The cell powder was either stored at -80°C or used directly for MNase digestion and RNA isolation.

Growth protocol

Arabidopsis cell line (Columbia ecotype, Col-0) was maintained in 250 mL Erlenmeyer flasks, filled with 50 mL of cell culture medium (Gamborg’s B5 basal medium with minor organic (Sigma G5893) in 1.1 mg/L 2,4-D, 3 mM MES and 3% sucrose)(described in Lee et al., 2010 and Tanurdzic et al., 2008). Cells were grown on a rotary shaker 160 rpm at 23°C (LS-X (Lab Shaker), Kuhner Shaker X). Constant light was used whereas dark grown cells were incubated under the same shaking and temperature condition but the flask were covered of aluminum foil. Cell line was subcultured every 7 days with a 2:50 (inoculum: fresh medium) dilution ratio. Cultures were carried out in duplicates.

Extracted molecule

total RNA

Extraction protocol

Chromatin Digestion
Pelleted cell culture was ground in a liquid nitrogen-cooled pestle and mortar to generate a cell powder. 1mL of the powder was suspended in a 0.5mL modified spheroplast digestion buffer & Nonidet P40 (SDBN: 1M sorbitol, 10mM NaCl, 50 mM TrisŸHCl pH7.5, 5mM MgCl2, 1 mM CaCl2, 1 mM 2-mercaptoethanol, 0.5 mM spermidine, 0.075% Nonidet P40). 300 µl of cells were transferred into 1.5mL microcentrifuge tube containing 30 or 120 units of MNase (Affimetrix). Cells were digested with MNase at 37°C for 3 min. The MNase digestion reactions were stopped by addition of and thorough mixing 30 µl of STOP solution containing 5% SDS and 250 µl EDTA. DNA was extracted by addition of an equal volume of 2X CTAB supplemented with PVP (200 mM Tris-HCl, 40 mM EDTA, pH 8.0; 2.8 M NaCl, 4% w/v CTAB, 2% PVP-40 cas number 9003-39-8) was added to the digestion reaction and incubated at 45°C for 15 mins. DNA was separated from protein by two phenol:chloroform (300:300) steps. DNA was precipitated with sodium acetate and propan-2-ol then washed in 80% ethanol and dried. Samples were incubated with 10X RNase A at 37°C for 1h with 100U unmodified T4 polynucleotide kinase (NEB) for further 30 min at 37°C.
MNase digested DNA was separated on 1.5% agarose gel, stained with ethidium bromide for 10 min at 80V, DNA fragments ranging from 20 bp to 1 kb were cut out and gel pieces containing DNA were placed in a Costar Spin-X 0.45 µm cellulose acetate centrifuge tube filter (Sigma CLS8136). Two series of freeze-thaws (-80C for 10 mins/RT for 10 mins) were performed to macerate the gel. Filter tubes were centrifuged twice (14kg; 5 min; RT) with a 180 degree rotation of the tubes between the two spins. DNA in the aqueous phase was then extracted with phenol:chloroform (200:200), and precipitated at -80°C for 30 mins with sodium acetate to propan-2-ol and finally washed with 80% ethanol before being resuspended in ultrapure milliQ water.
RNA isolation
Total RNA was extracted using the RNeasy Plant mini Kit (Qiagen, http://www.qiagen.com).
Nucleic acid quantification and quality check
DNA samples were quantified on QubitTM 2.0 Fluorometer with dsDNA BR assay according to manufacturer’s instructions.
RNA was quantified using QubitTM 2.0 fluorometer with Qubit RNA BR assay according to manufacturer’s instructions. RNA quality was checked on 1.5% agarose gel.
Exeter Sequencing Service and Computational core facilities at the University of Exeter performed the Genomic and RNA library preparation, quantification and 50bp paired end sequencing on HiSeq 2500.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2500

Description

Dark-RNA3
processed data column: ES06

Data processing

Alignment: bowtie allowing 3 base missmatches against TAIR10 genome from paired-end input
Total coverage: Joined paired reads with bamToBed -bedpe
Feature alignments: danpos.py to manual specification
RNAseq: tophat2 with Araport11.gtf annotation and TAIR10.fasta reference genome
Isoform Analysis: cufflinks with Araport11.gtf and TAIR10.fasta
Quantification and Differential Expression analysis: HTseq and edgeR (Anders, S. et. al., Bioinformatics, 2015)(Robinson et al., Bioinformatics, 2009)
Genome_build: Athal Col0 TAIR10
Supplementary_files_format_and_content: Wig.gz: Fixed width genomic traces of MNase digested samples at midpoints or total coverage at size separation; *csv: Rawcounts and edgeR output

Submission date

Feb 01, 2017

Last update date

May 15, 2019

Contact name

Daniel Antony Pass

Organization name

Cardiff University

Street address

Museum Avenue

City

Cardiff