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| Status |
Public on Jul 11, 2016 |
| Title |
met1_epiRIL_01_MethylC-seq |
| Sample type |
SRA |
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| Source name |
met1_epiRIL_01_MethylC-seq
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| Organism |
Arabidopsis thaliana |
| Characteristics |
accession: met1 epiRIL 01
tissue: leaf
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| Growth protocol |
All plants with the exception of met1/sdg7/sdg8 mutants and their controls were sown onto soil and grown in growth chamber under 18 hr light, 6 hr dark at 23-25C. met1/sdg7/sdg8 mutants and their wild-type controls were sown and germinated on MS media.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Tissue was flash frozen in liquid nitrogen for all experiments including ChIP-seq, RNA-seq and MethylC-seq. DNA was isolated using a Qiagen Plant DNeasy kit (Qiagen, Valencia, CA) following the manufacturer’s recommendations. RNA was isolated using TRIzol (Thermo Scientific, Waltham, MA) following the manufacturer’s instructions
MethylC-seq libraries were constructed using the MethylC-seq protocol (Urich et al. 2015).
RNA-seq: RNA-seq libraries were constructed using Illumina TruSeq Stranded RNA LT Kit (Illumina, San Diego, CA) following the manufacturer’s instructions with limited modifications. The starting quantity of total RNA was adjusted to 1.3 µg, and all volumes were reduced to a third of the described quantity.
CHIP-seq: Immunoprecipitated DNA was end repaired using the End-It DNA Repair Kit (Epicentre, Madison, WI) according to the manufacturer’s instructions. DNA was purified using Sera-Mag (Thermo Scientific, Waltham, MA) at a 1:1 DNA to beads ratio. The reaction was then incubated for 10 minutes at room temperature, placed on a magnet to immobilize the beads, and the supernatant was removed. The samples were washed two times with 500µL of 80% ethanol, air dried at 37C and then resuspended in 50ul of 10 mM Tris-Cl pH8.0. Finally, the samples were incubated at room temperature for 10 minutes, placed on the magnet, and the supernatant was transferred to a new tube, which contained reagents for “A-tailing.” A-tailing reactions were performed at 37C according to the manufacturers instructions (New England Biolabs, Ipswich, MA). The samples were cleaned using Sera-Mag beads as previously described. Next, adapter ligation was performed using Illumina Truseq Universal Y-adapters and T4 DNA ligase (New England Biolabs, Ipswich, MA) overnight at 16C. A double clean-up using the Sera-Mag beads was performed to remove any adapter-adapter dimers and the elution was used for 15 rounds of PCR. Lastly, samples were cleaned up one final time using the procedures described above.
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| Library strategy |
Bisulfite-Seq |
| Library source |
genomic |
| Library selection |
RANDOM |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
Whole Genome Bisulphite Sequencing of A. thaliana met1 epiRIL line 1
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| Data processing |
For MethylC-seq data: Reads were trimmed, aligned, and methylation called using the methylpy pipeline (Schultz et al. 2015). The genome is converted into a forward strand reference (all Cs to Ts) and a reverse strand reference (all Gs to As). Cutadapt (Martin et al. 2011) is used to trim adaptor sequences and then bowtie (Langmead et al. 2009) aligns reads to the two converted reference genomes. Only uniquely aligned reads are retained and the non-conversion rate calculated from unmethylated reads aligned to the chloroplast genome or spiked in unmethylated lambda DNA.
For RNA-seq data: Raw FASTQ reads were trimmed for adapters, preprocessed to remove low quality reads using Trimmomatic v0.32 (Bolger et al. 2014). Reads were aligned using Tophat v2.0.13 (Trapnell et al. 2010) supplied with a reference GFF file and the following arguments: -I 50000 --b2-very-sensitive --b2-D 50. Transcripts were then quantified using Cufflinks v2.2.1 (Trapnell et al. 2012) supplied with a reference GFF file.
For CHIP-seq data: Raw ChIP reads were trimmed for adapters and low-quality bases using Trimmomatic version 0.32 (Bolger et al. 2014). Reads were trimmed for TruSeq version 3 single-end adapters with maximum of two seed mismatches, palindrome clip threshold of 30 and simple clip threshold of 10. Additionally, leading and trailing bases with quality less than 10 were removed; reads shorter than 50 bp were discarded. Trimmed reads were mapped to the TAIR10 genome using bowtie2 version 2.2.3 (Langmead et al. 2012) with default options. Mapped reads were sorted using samtools verison 1.2 (Li et al. 2009) then clonal duplicates were removed using samtools version 0.1.9 (Li et al. 2009). BAM files were then converted to BED files using bedtools v2.21.1 (Quinlan and Hall 2010).
Genome_build: A. thaliana = TAIR10; A. lyrata = v1.0; B. rapa = FPsc v1.3 (http://phytozome.jgi.doe.gov/pz/portal.html#!info?alias=Org_BrapaFPsc); B. oleracea = TO1000 v1.0 (GCF_000695525.1); C. rubella = v1.0 (GCF_000375325.1); E. salsugineum = v1.0 (GCF_000478725.1)
Supplementary_files_format_and_content: MethylC-seq data (marked as “allc_total.tsv”) column 1 = chr = chromosome; column 2 = pos = coordinate for the cytosine position on the chromosome; column 3 = strand = + or - strand; column 4 = mc_class = context of the cytosine and the two following bases from the same strand; column 5 = mc_count = number of reads supporting a methylated cytosine; column 6 = total = total number of reads at that position; column 7 = methylated = cytosine is considered methylated if there is a 1 or unmethylated if there is a 0
Supplementary_files_format_and_content: RNAseq data column 1 = gene_ID = gene identifier; column 2 = FPKM = gene expression in fragments per kilobase per million (FPKM)
Supplementary_files_format_and_content: BED file of H2A.Z CHIP-Seq data
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| Submission date |
Nov 17, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Robert J Schmitz |
| E-mail(s) |
schmitz@uga.edu
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| Organization name |
University of Georgia
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| Department |
Genetics
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| Street address |
B416 Davison Life Sciences
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| City |
Athens |
| State/province |
GA |
| ZIP/Postal code |
30602 |
| Country |
USA |
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| Platform ID |
GPL19580 |
| Series (1) |
| GSE75071 |
On the Origin and Evolutionary Consequences of Gene Body DNA Methylation |
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| Relations |
| BioSample |
SAMN04276619 |
| SRA |
SRX1436245 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1942131_A_thaliana_met_epiRIL_01_allc_total.tsv.gz |
161.9 Mb |
(ftp)(http) |
TSV |
SRA Run Selector |
| Processed data provided as supplementary file |
| Raw data are available in SRA |
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