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| Status |
Public on Oct 12, 2023 |
| Title |
L18-SR |
| Sample type |
SRA |
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| Source name |
Triticum aestivum 14-days-old seedlings
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| Organism |
Triticum aestivum |
| Characteristics |
tissue: 14-days-old seedlings
cultivar: JW1
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| Treatment protocol |
We collected fresh urediospores of Pst race CYR32 from the leaves of wheat cultivar MingXian169. We dispersed the fresh spores in tap water and spread equal contents on the second leaf of control and transgenic wheat plants. The infected plants were incubated in a humid chamber overnight at 16 °C in dark. We transferred the infected plants to a growth chamber (16 °C at 16 h light/8 h dark, 80 % RH) to allow the growth of rust pathogens.
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| Growth protocol |
Common wheat (Triticum aestivum variety JW1) seeds were surface-sterilized via a 10-min incubation in 30% H2O2 and then thoroughly washed five times with distilled water. The seeds were germinated in water for 3 days at 22 °C, after which the germinated seeds with residual endosperm were transferred to soil. The seedlings were cultivated under long-day conditions.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Chromatin or RNA was extracted from 14-days-old seedlingss, using standard protocols.
For RNA-seq, library construction and deep sequencing were performed by Novogene (Beijing, China) using Illumina NovaSeq 6000 system to produce 150-bp paired-end reads.
For ChIP-seq, crosslinked materials were ground into fine power with liquid nitrogen and resuspended in ChIP Lysis Buffer1 (CLB1: 50 mM HEPES [pH 7.5], 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 10% Glycerol, 1x inhibitor Cocktail, 0.035% 2-Mercaptoethanol) and incubated for 60 minutes with rotation at 4°C. After incubation, collect the nucleus after filtering the mixture through 40-um strainer, centrifuging at 3000 g for 30 minutes at 4°C in a swinging bucket rotor and removing the supernatant. The nucleus was washed twice with ChIP Lysis Buffer2 (CLB2: 50 mM HEPES [pH 7.5], 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 10% Glycerol, 1x inhibitor Cocktail). DNA was sheared by sonication to approximately 300–500-bp fragments. After centrifugation (10 minutes at 13,000 rpm), the supernatant was precleared with 40 μl salmon sperm (SS) DNA/Protein A agarose for 60 minutes at 4°C. After 2 minutes of centrifugation at 500 g, the supernatant was transferred to a siliconized tube, and 10 μl of the appropriate antibody (H3 trimethyl-Lys 27 (ABclonal, A2363), H3 trimethyl-Lys 4 (Abcam, Cambridge, England), and H3 acetyl-Lys 9 (Millipore)) was added. After incubation overnight with rotation, 40 μl SS DNA/Protein A agarose was added and incubation continued for 1 hr. The agarose beads were then washed with 1 ml of each of the following: Low salt buffer (50 mM HEPES [pH 7.5], 150 mM NaCl, 1 mM EDTA), High salt buffer (50 mM HEPES [pH 7.5], 635 mM NaCl, 1 mM EDTA), LiCl wash buffer (0.25M LiCl, 0.5% NP-40, Tris-HCl [pH 8], 1 mM EDTA), and 1× TE (10 mM Tris-HCl [pH 8], 1 mM EDTA). The immunocomplexes were eluted from the beads with 400 μl 1% SDS, 0.1 M NaHCO3. A total of 20 μl 5 M NaCl was then added to each tube, and crosslinks were reversed by incubation at 65°C for 5–6 hours. Residual protein was degraded by the addition of 20 μg Prot K (in 10 mM EDTA and 40 mM Tris [pH 8.0]) at 45°C for 1 hour, followed by phenol/chloroform/isoamyl alcohol extraction and ethanol precipitation. Pellets were washed with 70% EtOH and resuspended in 30 μl TE buffer. More than 10 ng ChIP DNA was used to prepare each sequencing sample. Libraries were constructed and sequenced by Novogene (Beijing, China). The libraries were sequenced with the Illumina NovaSeq 6000 system to produce 150-bp paired-end reads.
For spike-in ChIP-seq, Arabidopsis chromatin was added into wheat chromatin before immunoprecipitation. Briefly, before performing immunoprecipitation, 40 ul of chromatin was reverse crosslinked, proteinase K digested followed by DNA purification. The concentration of purified DNA was determined using Qubit dsDNA High-Sensitivity Assays (Invitrogen). Meanwhile, chromatins from Arabidopsis were also isolated and determined the concentration with the same method. One percent of Arabidopsis chromatins were mixed with different Wheat samples before immunoprecipitation. The rest procedures are the same as the previous description.
For nascent RNA (chromatin-bound RNA, CB RNA) library preparation, the chromatin RNA extraction was completed as previously described54. After degrading genomic DNA by TURBO DNase (Life Technologies), CB RNA was subjected to rRNA depletion using a riboPOOL kit (siTOOLs Biotech, PanPlant-10 nmol) and polyA RNA removal by oligo(dT) beads (NEB, S1419). Both CB RNA and polyA RNA were transformed into cDNA libraries using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (NEB #E7765) and sequenced on an Illumina NovaSeq platform.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
Sequencing reads were cleaned with the fastp (version 0.20.0)55, which eliminated bases with low quality scores (< 25) and irregular GC contents, sequencing adapters, and short reads. The remaining cleaned reads were mapped to the International Wheat Genome Sequencing Consortium (IWGSC) reference sequence (version 1.0) with the Burrows–Wheeler Aligner (version 0.7.17-r1188)56 for the wheat ChIP-Sequencing. For spike-in ChIP-seq, cleaned reads were mapped to the merged genome of common wheat (IWGSC) and Arabidopsis thaliana (TAIR10). The HISAT2 program (version 2.2.1)57 was used for mapping the RNA sequencing (RNA-seq) and chromatin-bound RNA (CB-seq) reads to the reference sequences. The MACS (version 2.2.6)58 program was used to identify the read-enriched regions (peaks) of the ChIP-Seq and CB-Seq data with the cutoff P < 1e−10. To quantify gene expression levels, the featureCount program of the Subread package (version 2.0.0)59 was used to determine the RNA-seq read density for the genes. To compare expression levels across samples and genes, the RNA-seq read density of each gene was normalized based on the exon length in the gene and the sequencing depth (i.e., fragments per kilobase of exon model per million mapped reads). To quantify histone markers across genes for the figure prepared with Integrative Genomics Viewer60, the number of reads at each position was normalized against the total number of reads (reads per million mapped reads). For spike-in ChIP-seq, the number of reads at each position was normalized against the total number of reads mapped to A. thaliana (reference-adjuseted reads per million, RRPM). The edgeR program61 was used for detecting differentially expressed genes based on the combined criteria: |log2 fold-change| > 1 and P < 0.05. The MAnorm2 package62 was used for the quantitative comparison of ChIP-Seq and CB-Seq signals between samples with the following criteria: |M value| > 1 and P < 0.05. For spike-in ChIP-seq, the value of RRPM was used to compare signals between samples with the following criteria: |log2 fold-change| > 1 and RRPM > 5. The peaks of CB-Seq overlapped with genes were identified as coding CB RNA. The peaks overlapped with 8 histone modifications (H3K27ac, H3K27me1, H3K27me3, H3K36me3, H3K4me1, H3K4me3, H3K9ac and H3K9me2) and not overlapped with genes were identified as enhancer RNA (eRNA). The remained CB RNA were identified as non-coding other CB RNA (nc-other). The genes with the peaks around their transcription start sites 3 kb were defined as eRNA targeted genes.
Assembly: IWGSC RefSeq v1.0
Supplementary files format and content: In ChIP-seq, peak files and bigwig files were provided; In mRNA-seq and CB-seq, RPKM for genes (IWGSC RefSeq v1.0) were provided.
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| Submission date |
Nov 22, 2022 |
| Last update date |
Oct 12, 2023 |
| Contact name |
yijing zhang |
| E-mail(s) |
zhangyijing@fudan.edu.cn
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| Organization name |
Fudan University
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| Department |
Biochemistry
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| Lab |
Functional Epigenomics Group
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| Street address |
2005 Songhu Road
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| City |
shanghai |
| ZIP/Postal code |
200438 |
| Country |
China |
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| Platform ID |
GPL25409 |
| Series (1) |
| GSE218538 |
LHP1-mediated conditional repression of subgenome-diversified defenses is responsible for the high plasticity of allopolyploid wheat |
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| Relations |
| BioSample |
SAMN31836754 |
| SRA |
SRX18348494 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6751453_L18-SR-V1.sort.q20.uniq.rpm.bw |
72.7 Mb |
(ftp)(http) |
BW |
| GSM6751453_featurecounts.L18-SR-V1.fpkm.txt.gz |
564.2 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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