 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Aug 21, 2017 |
| Title |
Mnase-seq of Arabidopsis thaliana: Light Grown High digest [Light-H1] |
| Sample type |
SRA |
| |
|
| Source name |
Mnase-seq_Light Grown High digest
|
| Organism |
Arabidopsis thaliana |
| Characteristics |
ecotype background: Col0
cell type: 16h post-stratification cell culture
growth condition: Light
mnase digestion: High
|
| 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 |
genomic DNA |
| 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 |
MNase-Seq |
| Library source |
genomic |
| Library selection |
MNase |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Description |
Light-H1
|
| 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 |
| State/province |
South Glamorgan |
| ZIP/Postal code |
CF10 3AX |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL17639 |
| Series (1) |
| GSE94377 |
Genome-wide chromatin mapping with size resolution reveals a dynamic sub-nucleosomal landscape in Arabidopsis |
|
| Relations |
| BioSample |
SAMN06288239 |
| SRA |
SRX2532083 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2474308_Light-H1_150-midpoints.wig.gz |
11.3 Mb |
(ftp)(http) |
WIG |
| GSM2474308_Light-H1_gt120.wig.gz |
19.2 Mb |
(ftp)(http) |
WIG |
| GSM2474308_Light-H1_lt120.wig.gz |
12.5 Mb |
(ftp)(http) |
WIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
|
|
|
|
 |
