|
| Status |
Public on Jun 04, 2021 |
| Title |
atxr5/atxr6 H3K26ac rep2 (ChIPseq) |
| Sample type |
SRA |
| |
|
| Source name |
3 old weeks leaves
|
| Organism |
Arabidopsis thaliana |
| Characteristics |
ecotype: Col0
tissue: leaves
age: 3 weeks
genotype: atxr5/atxr6 mutant
chip antibody: Active Motif: 39379
|
| Growth protocol |
Arabidopsis plants were grown under cool‐white fluorescent lights (approximately 100 μmol m−2 s−1) in long‐day conditions (16 h light/8 h dark).
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Leaves from three-week-old plants were fixed in 1% formaldehyde. Immunoprecipitation was performed using protein A magnetic beads (New England BioLabs). Following the Proteinase K treatment of each sample, immunoprecipitated DNA was purified using ChIP DNA Clean & Concentrator kit (Zymo Research, Irvine, CA, USA). 2 μl of Histone H3 antibody (Abcam: ab1791), 2.5 μl of H3K27me1 antibody (Millipore: 07-448), 2 ul of H3K27me2 antibody (Abcam: ab24684) or 2.5 μl of H3K27Ac antibody (Active Motif: 39135), was used per immunoprecipitation. For the ChIP experiments for H3K27ac, ChIP with exogenous genome (ChIP-Rx) was performed in order to properly normalize the data (Orlando et al, 2014). For each sample, an equal amount of drosophila chromatin (Active Motif #53083) was added prior to chromatin shearing.
ChIP library preparation was performed using TruSeq Library Prep Kit (Illumina). Libraries were validated using Agilent Bioanalyzer 2100 Hisense DNA assay and quantified using the KAPA Library Quantification Kit for Illumina® Platforms kit. Sequencing was done on an Illumina NovaSeq 6000 using the S4 XP workflow.
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| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Description |
ChIP with exogenous genome (ChIP-Rx) was performed in order to properly normalize the data (Active Motif #53083).
A56_36_rep2.norm.bw
|
| Data processing |
RNA-seq processing and Analysis:Two independent biological replicates for Col, atxr5/6, gcn5 and atxr5/6 gcn5 were sequenced. Paired-end reads were filtered and trimmed using BBTools (version 38.79). Reads with quality inferior to 20 were removed. The resulting data sets were aligned against the Arabidopis genome (TAIR10) using STAR (version 2.7.2a) allowing 2 mismatches (--outFilterMismatchNmax 2). Protein-coding genes and transposable elements (TE) were defined as described in the TAIR10 annotation gff3 file. The program featureCounts (version 1.6.4) (12) was used to count the paired-end fragments overlapping with the annotated protein-coding genes and TEs. Differential expression analysis of protein-coding genes was performed using DESeq2 version 1.26 on raw read counts to obtain normalized fold changes (FC) and Padj-values for each gene. Genes were considered to be differentially expressed only if they showed a log2FC >1 and a Padj-values < 0.05. TPM (transcripts per million) values were calculated for TEs. To define TEs as up-regulated in the atxr5/6 mutant, they must show 2-fold up-regulation as compared to Col in both biological replicates and have a value of TPM > 5.
ChIP-seq processing and Analysis: Paired-end reads were filtered and trimmed using BBTools . Reads with quality inferior to 20 were removed. Data sets were aligned against combined genomes of Arabidopsis thaliana (TAIR10) and Drosophila melanogaster (dm6) using bowtie2 with default parameters. Duplicate reads were removed using Picard toolkit (MarkesDuplicates with REMOVE_DUPLICATES=true). To calculate the Rx scaling factor of each biological replicate, Drosophila-derived IP read counts were normalized according to the number of input reads. Spike-in normalization was performed as previously described in Nassrallah et al. 2018. Bigwig file were then scaled by adjusting the number of reads in each bin with the Rx factors and therefore generating reference-adjusted reads per million (RRPM).
Genome_build: RNA-seq: TAIR10 ChIP-seq: TAIR10 and dm6 (ChIP-Rx)
Supplementary_files_format_and_content: RNA-seq: tab-delimited text files include RPKM values for each Sample was computed with featureCounts. ChIP-seq: bigwig files were generated using the H3k27ac or H3K36ac signal normalized with the Rx factor.
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| |
|
| Submission date |
Jul 15, 2020 |
| Last update date |
Jun 04, 2021 |
| Contact name |
Yannick Jacob |
| E-mail(s) |
yannick.jacob@yale.edu
|
| Phone |
203-432-8908
|
| Organization name |
Yale university
|
| Department |
Department of Molecular, Cellular & Developmental Biology
|
| Lab |
Jacob Lab
|
| Street address |
YSB 416 260 Whitney Avenue
|
| City |
New Haven |
| State/province |
Connecticut |
| ZIP/Postal code |
06511 |
| Country |
USA |
| |
|
| Platform ID |
GPL26208 |
| Series (1) |
| GSE146126 |
H3.1K27me1 maintains transcriptional silencing and genome stability by preventing GCN5-mediated histone acetylation |
|
| Relations |
| BioSample |
SAMN15543010 |
| SRA |
SRX8736790 |
