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Sample GSM2027815 Query DataSets for GSM2027815
Status Public on Jul 11, 2016
Title A_thaliana_H3K9me2_chip
Sample type SRA
 
Source name A. thaliana H3K9me2 Chip
Organism Arabidopsis thaliana
Characteristics accession: Col-0
tissue: leaf
chip antibody: H3K9me2
chip antibody vendor: Cell signalling
chip antibody cat. #: 9753s
chip antibody lot #: 2
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.
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).
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.
 
Library strategy ChIP-Seq
Library source genomic
Library selection ChIP
Instrument model Illumina NextSeq 500
 
Description ChIP seq data for A. thaliana H3K9me2
Data processing 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; B. oleracea = TO1000 v1.0; C. rubella = v1.0; E. salsugineum = v1.0
Supplementary_files_format_and_content: BED file of CHIP-Seq data
 
Submission date Jan 05, 2016
Last update date May 15, 2019
Contact name Robert J Schmitz
E-mail(s) schmitz@uga.edu
Organization name University of Georgia
Department Genetics
Street address B416 Davison Life Sciences
City Athens
State/province GA
ZIP/Postal code 30602
Country USA
 
Platform ID GPL19580
Series (1)
GSE75071 On the Origin and Evolutionary Consequences of Gene Body DNA Methylation
Relations
BioSample SAMN04386104
SRA SRX1518742

Supplementary file Size Download File type/resource
GSM2027815_A_thaliana_H3K9me2_chip.bed.gz 222.4 Mb (ftp)(http) BED
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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