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Sample GSM6133432

Query DataSets for GSM6133432

Status

Public on Mar 16, 2023

Title

RNA-seq, vel3, 24HAI, R2

Sample type

SRA

Source name

water-imbibed endosperm

Organism

Arabidopsis thaliana

Characteristics

tissue: water-imbibed endosperm
genotype: vel3

Extracted molecule

polyA RNA

Extraction protocol

RNA of endosperm and seeds were extracted using Qiagen RNeasy Plant Mini Kit
Libraries of RNA-seq and H3K27me3 ChIP-seq were construed by Novogene (China) and BGI (China) respectively. For libraries of VEL3 ChIP-seq, Tn5 tagmentation was performed on 1ng ChIP-ed DNA or input DNA for 5 min at 55°C, with 0.1ul enzyme and 2.5ul buffer in 5ul reaction (Illumina, 20034197). PCR amplification was conducted using the following programs: 72°C 5min, 98°C 30s, N cycles of 98°C 10s, 63°C 30s and 72°C 1min. cycle number (N) was determined by the qpcr of each partially-amplified library. 0.8X Agencourt AMPure XP beads (Beckman Coulter, A63880) was used for the library size selection. Libraries were sequenced at Novogene (Beijing, China) using a Novaseq system, 150-bp paired-end sequences with a minimum of 30 million reads acquired per sample.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 4000

Description

24HAI_endosperm_TPM
vel3_R2

Data processing

For RNA-seq, raw reads were trimmed using cutadapt-1.9.1 (Martin, 2011) and mapped to Arabidopsis thaliana TAIR10 reference genome using STAR-2.5.a (Dobin et al., 2013). featureCounts (Liao et al., 2014) was used to count the numbers of reads mapped to each gene. Sense reads were selected for downstream analysis. edgeR (Robinson et al., 2010) was used to calculate differentially expressed genes (DEGs), based on the threshold of FDR<0.05 and fold change≥2. For data visualization, bigwig files were generated using deepTools-3.1.1 (Ramírez et al., 2016) with a bin size of 50bp, before visualization in IGV -2.12.3 (Robinson et al., 2011). For ChIP-seq, raw reads were trimmed using cutadapt-1.9.1 (Martin, 2011) and mapped to Arabidopsis thaliana TAIR10 reference genome using bowtie2 (Langmead et al., 2018). Only uniquely mapped reads were kept for downstream analysis using Samtools-1.9 and Sambamba-6.7 (Li et al., 2009; Tarasov et al., 2015). For data visualization, bigwig files were calculated using deepTools-3.1.1(Ramírez et al., 2016) with a bin size of 50bp, before visualization in IGV -2.12.3 (Robinson et al., 2011). Peaks were called using SICER 1.1 for H3K27me3 (Zhang et al., 2009) or MACS2 for VEL3 (Zhang et al., 2008), based on the threshold of FDR<0.05. Only common peaks between two biological replicates were considered. Different H3K27me3 peaks between Col0 and vel3 were calculated using DiffBind (Ross-Innes et al.,2012) and DESeq2 (Love et al., 2014), based on the threshold of FDR<0.05 and fold change≥2. Genes were identified for these peaks are located in genebody or 1kb promoter using bedtools-2.28.0 (Quinlan & Hall, 2010).
Supplementary files format and content: For RNA-seq, csv files with TPM are provided. For ChIP-seq, bigwig files are provided

Submission date

May 11, 2022

Last update date

Mar 16, 2023

Contact name

Xiaochao Chen

E-mail(s)

xiaochao.chen@jic.ac.uk

Organization name

John Innes Centre

Department

Crop Genetics