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Sample GSM6622813 Query DataSets for GSM6622813
Status Public on Feb 23, 2024
Title RNA_Col_rep2
Sample type SRA
 
Source name 5-day-old seedlings
Organism Arabidopsis thaliana
Characteristics tissue: 5-day-old seedlings
genotype: Col
Growth protocol Seeds were sown in soil and stratified for 2 days at 4°C. The plants were then grown in a growth chamber with a photoperiod of 16 h of light and 8 h of darkness at 22°C.
Extracted molecule total RNA
Extraction protocol Total RNA from 5-day seedling was extracted with a RNeasy Plant Mini kit (Qiagen). For RNA-seq analysis, the total RNA was treated with a Poly(A) mRNA Magnetic Isolation Module Kit (NEB #E7490). DNA of 6th, 7th and 8th leaves was purified using a DNeasy® Plant Mini Kit (Qiagen) and eluted in 150 µL of elution buffer. Experiments were performed for two biological replicates. The nuclear matrix was isolated as previously described in (53). Briefly, Arabidopsis 5-day-old whole plants grown under LD condition (16h at 23 °C under 22 μmol·m−2·s−1 continuous white light, 8h at 23 °C dark with 70% humidity) were collected and chopped in 1 ml LB01 buffer (2 mM Na2EDTA, 20 mM NaCl, 2 mM EDTA, 80 mM KCl, 0.5 mM spermine, 15 mM β-mercaptoethanol, 0.1% Triton X-100, pH 7.5). Another 9 ml LB01 buffer was used to wash the nuclei into 1-layer miracloth filter followed by a one-time filter through 30 μm CellTrics (Sysmex, Germany, 04-0042-2316). The nuclei mixture was centrifuged at 1500g for 10 min at 4 °C. The nuclei pellet was further wash with DNase I buffer (20 mM Tris pH 7.4, 20 mM KCl, 70 mM NaCl, 10 mM MgCl2, 0.125 mM spermidine 1 mM PMSF, 0.5% Triton-X 100) until white. After centrifuge, the nuclei were resuspended in 200 μl DNase I buffer, and an aliquot of nuclei was stored for isolation and estimation of total genomic DNA as control. DNase I (Thermo Scientific, EN0525) was added and incubated at 4°C for 1 hr. Nuclei were collected by centrifugation at 3000g for 10 min at 4 °C. Digestion was followed by extraction with 0.4 M NaCl for 5 min twice on ice in Extraction Buffer (10 mM Hepes pH 7.5, 4 mM EDTA, 0.25 mM spermidine, 0.1 mM PMSF, 0.5% (v/v) Triton X-100). Another two times extraction with 2 M NaCl for 5 min on ice in Extraction Buffer. The final nuclear matrix pellet was washed twice with Wash Buffer (5 mM Tris pH 7.4, 20 mM KCl, 1 mM EDTA, 0.25 mM spermidine, 0.1 mM PMSF).
RNA-seq Libraries were prepared from the resulting mRNA with a NEBNext Ultra Kit (NEB #E7530). The libraries were constructed according to a standard protocol (Illumina). The RNA-seq libraries were sequenced at Novogene via a Novaseq instrument in 150-bp paired-end mode. MAR-seq libraries were prepared using NEBNext® Ultra™ II DNA Library Prep Kit (NEB, E7645) according to the manufacturer’s instructions. The libraries were sequenced at Novogene (UK) via a Novaseq instrument in 150-bp paired-end mode. Two biological replicates were performed.
 
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina NovaSeq 6000
 
Description 2x
Data processing For RNA-seq analysis, reads were mapped to TAIR10 Col-0 and the Ler‐0 genomes with HISAT2 (Kim et al., 2019) in paired-end mode.
DEGs were analyzed via the Subread (Liao et al., 2019) and DESeq2 R packages (Love et al., 2014) with a 0.05 false discovery rate (FDR).
For MAR-seq analysis, quality control and adapter trimming of MAR-seq reads were performed with Fastp (61). Reads were aligned to the TAIR10 reference genome using Bowtie2. Mapped reads were deduplicated using MarkDuplicates (https://broadinstitute.github.io/picard). Coverage was estimated and normalized to 10 million reads. MAR-seq peaks in wild-type and mutants were called by SCIER2 (62).
Assembly: tair10
Supplementary files format and content: bedGraph, counts
 
Submission date Oct 10, 2022
Last update date Feb 23, 2024
Contact name Hua Jiang
E-mail(s) jiangh@ipk-gatersleben.de
Phone 049394825875
Organization name Leibniz Institute of Plant Genetics and Crop Plant Research (IPK) Gatersleben
Lab Group of Applied Chromosome Biology
Street address Corrensstraße 3
City Gatersleben, Stadt Seeland, Germany
ZIP/Postal code 06466
Country Germany
 
Platform ID GPL26208
Series (1)
GSE215135 Chromatin attachment to the nuclear matrix represses hypocotyl elongation in Arabidopsis thaliana
Relations
BioSample SAMN31228301
SRA SRX17839704

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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