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Status |
Public on Nov 30, 2023 |
Title |
ribotag blue WT replicate 3 |
Sample type |
SRA |
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Source name |
6-days-old-seedling
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Organism |
Arabidopsis thaliana |
Characteristics |
tissue: 6-days-old-seedling chip antibody: Anti-FLAG M2 genotype: WT ecotype: Col-0
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Growth protocol |
A light-emitting diode was used to generate monochromatic blue light (peak 450 nm; half-bandwidth of 20 nm) and cool white fluorescent tubeswere used for white light. The Arabidopsis seedlings used in these experiments were grown in either a growth chamber (Conviron, model no. E7/2) or growth room at 21 °C under different light regimes.
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Extracted molecule |
total RNA |
Extraction protocol |
TRAP of polysomes was performed as described previously (Mustroph et al., 2009). Briefly, 1 mL of pulverized tissue was added to a 15 mL tube containing 5 mL of polysome extraction buffer (PEB: 200 mM Tris, pH 9.0, 200 mM KCl, 25 mM EGTA, 35 mM MgCl2, 1% PTE, 1 mM DTT, 1 mM PMSF, 100 μg/mL cycloheximide, 50 μg/mL chloramphenicol) and 1% detergent mix [20% (w/v) polyoxyethylene(23), 20% (v/v) Triton X-100, 20% (v/v) Octylphenyl-polyethylene glycol, 20% (v/v) Polyoxyethylene sorbitan monolaurate 20] and incubated until thawed on ice, before homogenized with a glass homogenizer. The homogenized mixture was incubated on ice for ten minutes before being centrifuged at 16,000g at 4 degrees Celsius for fifteen minutes. After centrifugation, the supernatant was transferred to a fresh tube and filtered through sterile Miracloth (Millipore) to create an extract that had been clarified. 10% of the total cleared extract was preserved to be used as an input for isolating RNA. Anti-FLAG M2 Protein beads (1.5 mL) that were pre-washed twice with wash buffer [WB; 200 mM Tris (pH 9.0), 200 mM KCl, 25 mM EGTA, 35 mM MgCl2, 1 mM DTT, 1 mM PMSF, 100 g/mL cycloheximide, 50 g/mL chloramphenicol] were added to the clar The binding of ribosome-mRNA complexes with a FLAG epitope tag was achieved by incubating them at 4°C for two hours while gently shaking the tubes. Remove the supernatant and carefully resuspend the beads in 6 mL WB for 2 minutes at 4°C while rocking. This procedure was performed twice more before the beads were resuspended in 1 mL WB. The beads were washed twice more with 1 mL WB, and then the supernatant was extracted. Added 300μL of WB with 200ng/μL of FLAG3 peptide and 20U/μL RNase inhibitor (Cat. # N8080119 Themo Fisher) and incubated for 30 mins at 4°C with shaking. Centrifuged and collected the supernatant for RNA purification. For meRIP, WT and mutant seedlings grown in the dark or in blue light (30 μmol m−2 s−1) for 6 days were used for m6A-seq.Total RNA was isolated using Direct-zol RNA Miniprep Kits (Zymo, Cat no. R2052). MeRIP-seq was performed as described previously (Wang et al., 2021).The libraries from two biological repeats for each sample were sequenced on Illumina Novaseq6000 instruments in pair-end mode with 100 bp per reads. The RNA was used for preparation of RNA-seq libraries with TruSeq RNA library prep kit (Illumina). The libraries from 3 biological repeats for each sample were sequenced on the Illumina HiSeq 2500 sequencing systems.
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Library strategy |
OTHER |
Library source |
transcriptomic |
Library selection |
other |
Instrument model |
Illumina NovaSeq 6000 |
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Data processing |
Cleaned reads of Ribo-tag and input samples were aligned to the TAIR10 reference genome with Bowtie2 (v2.1.0) (Langmead and Salzberg, 2012). Translation efficiency abundance was measured by RSEM using the default parameters (Li and Dewey, 2011).Translation efficiency was estimated with (RPKM in Ribotag +1)/ (RPKM in input +1). Differential translation efficiency analysis was conducted using edgeR (Robinson et al., 2010)with a threshold of p-value < 0.05 and Fold Change > 1.5 was used to determine whether there were any significant differences in translation efficiency between samples. The adapter sequence of m6A meRIP raw reads were trimmed by trim_galore (https://www.bioinformatics.babraham.ac.uk/projects/trim_galore/). The trimmed reads were aligned to the TAIR10 reference genome with Bowtie2 (Langmead and Salzberg, 2012) (v2.1.0) with default settings. meRIP track files in BigWig format were generated using bamCoverage of deeptools (v3.1.3) with RPKM normalization (Ramirez et al., 2016) from de-duplicated reads of Samtools (Li et al., 2009). m6A peaks were called by MACS2 (v2.1.1) and annotated using ChIPseeker (Yu et al., 2015; Zhang et al., 2008). Differential peaks were called with a threshold of p-value < 0.05 and Fold Change > 1.5. m6A data metaplots were plotted by deeptools (v2.5.1) (Ramirez et al., 2014). Assembly: TAIR10 Supplementary files format and content: bigwig: meRIP intensity. Supplementary files format and content: *RPKM.txt: signal intensity of m6A peaks Supplementary files format and content: *results: gene expression level Library strategy: Ribo-tag
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Submission date |
Mar 11, 2023 |
Last update date |
Nov 30, 2023 |
Contact name |
Zhenhui Zhong |
E-mail(s) |
zhenhuizhong@gmail.com
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Organization name |
University of California, Los Angeles
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Department |
Department of Molecular, Cell and Developmental Biology
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Lab |
Jacobsen Lab
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Street address |
610 Charles E Young Dr East
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City |
Los Angeles |
ZIP/Postal code |
90095 |
Country |
USA |
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Platform ID |
GPL26208 |
Series (1) |
GSE227150 |
Light-induced LLPS of the CRY2/SPA1/FIO1 complex regulating mRNA methylation and chlorophyll homeostasis in Arabidopsis |
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Relations |
BioSample |
SAMN33725540 |
SRA |
SRX19638855 |
Supplementary file |
Size |
Download |
File type/resource |
GSM7092449_5-WT-3.genes.results.gz |
857.4 Kb |
(ftp)(http) |
RESULTS |
SRA Run Selector |
Raw data are available in SRA |
Processed data provided as supplementary file |
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