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| Status |
Public on Nov 30, 2016 |
| Title |
CC DME |
| Sample type |
SRA |
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| Source name |
central cell
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| Organism |
Arabidopsis thaliana |
| Characteristics |
tissue: central cell
genotype/variation: DEMETER loss of function mutant
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| Growth protocol |
A. thaliana plants homozygous for the DD7:NTF transgene were grown on soil in either a greenhouse or in an environment-controlled room with a long-day photoperiod (16 hr light, 8 hr dark).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
We emasculated stage 12 A. thaliana flowers, which have yellow anthers that have not dehisced. The emasculated plants were then incubated in an environment-controlled room or in a growth chamber with a long-day photoperiod for 24 hours. Pistils were dissected, opened, and ovules exposed to a protoplasting enzyme solution in a vacuum based on methods described in (Yoo et al. 2007 Nature Protocols 2:1565-1572). Protoplasts were gently pelleted by centrifugation, the supernatant was removed, and the pellet was resuspended in protoplast lysis buffer adapted from (Sheen 1993 Embo Journal 12:3497-3505). Modified procedures based on the INTACT method (Deal and Henikoff 2011 Nat Protoc 6:56-68) were used to purify central cell nuclei. To remove cell debris, samples were pre-incubated with Dynal Protein-G magnetic beads (Invitrogen: 100-03D), which were pelleted by exposure to a magnetic field. The supernatant was removed and incubated with anti-GFP antibodies (Invitrogen: G10362), which were precipitated with Protein-G magnetic beads. Central cell nuclei were dissociated from the anti-GFP beads and stored at -80C.
Bisulfite sequencing libraries were constructed as described in (Smallwood et al. 2014 Nat Methods 11:817-820). We followed the single-cell library preparation protocol using Klenow exo- from Enzymatics (Beverly, MA, USA). Library amplification was performed with 13 PCR cycles using the EPIK Amplification Kit (Bioline GmbH, Germany).
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| Library strategy |
Bisulfite-Seq |
| Library source |
genomic |
| Library selection |
RANDOM |
| Instrument model |
Illumina HiSeq 4000 |
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| Data processing |
Raw reads were trimmed using trim galore (http://www.bioinformatics.babraham.ac.uk/projects/trim_galore) with the --clip_R1 9 parameter
Trimmed reads were aligned to their respective reference genomes (TAIR10/MSU7 including mitochondira and chloroplast contigs) using Bismark (http://www.bioinformatics.babraham.ac.uk/projects/bismark/) with the --non_directional parameter
CG/CHG/CHH Methylation was extracted from the bismark BAM file using the bismark_methylation_extractor command using the following parameters; --comprehensive --cytosine_report --bedGraph --CX
Single_C: We used a Perl script to convert the extracted methylation from bismark_methylation_extrator into single-c files for each sequence context (CG, CHG, CHH)
50bp_window: We used a Perl script to calculate fractional methylation (#C/(#C+#T)) within a 50 bp sliding window for each sequence context (CG, CHG, CHH).
Genome_build: MSU7 (Oryza sativa)
Genome_build: TAIR10 (Arabidopsis thaliana)
Supplementary_files_format_and_content: All files are in GFF format. Files contain fractional methylation data either for individual cytosines (single-c) or in 50 bp windows.
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| Submission date |
Nov 13, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Xiaoqi Feng |
| E-mail(s) |
xiaoqi.feng@jic.ac.uk
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| Organization name |
John Innes Centre
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| Department |
Cell and Developmental Biology
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| Lab |
Xiaoqi Feng
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| Street address |
Norwich Research Park
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| City |
Norwich |
| State/province |
Norfolk |
| ZIP/Postal code |
NR4 7UH |
| Country |
United Kingdom |
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| Platform ID |
GPL21785 |
| Series (1) |
| GSE89789 |
DNA demethylation is initiated in the central cells of Arabidopsis and rice |
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| Relations |
| BioSample |
SAMN06013831 |
| SRA |
SRX2343949 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2388876_CC_DME_B4_At.CG.w1.gff.gz |
33.5 Mb |
(ftp)(http) |
GFF |
| GSM2388876_CC_DME_B4_At.CG.w50.gff.gz |
13.8 Mb |
(ftp)(http) |
GFF |
| GSM2388876_CC_DME_B4_At.CHG.w1.gff.gz |
37.4 Mb |
(ftp)(http) |
GFF |
| GSM2388876_CC_DME_B4_At.CHG.w50.gff.gz |
15.4 Mb |
(ftp)(http) |
GFF |
| GSM2388876_CC_DME_B4_At.CHH.w1.gff.gz |
151.1 Mb |
(ftp)(http) |
GFF |
| GSM2388876_CC_DME_B4_At.CHH.w50.gff.gz |
24.4 Mb |
(ftp)(http) |
GFF |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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