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Sample GSM4057573

Query DataSets for GSM4057573

Status

Public on Feb 01, 2020

Title

RNA-seq replicate2 of CR stage 2

Sample type

SRA

Source name

rice crown root initials

Organism

Oryza sativa Japonica Group

Characteristics

tissue: stage 2 of crown root initials
genotype/variation: Hwayoung cultivar

Growth protocol

Rice (Oryza sativa spp japonica) material used in this study is from ‘Hwayoung’ (HY) background. For germination culture, seeds were surface-sterilized and germinated in medium containing 0.8% agar supplemented with 3% (w/v) sucrose at 28°C (in light) and 24°C (in dark) with a 14-h-light/10-h-dark cycle. For growth in field, germinated rice seedling was transferred to greenhouse at 25–30 °C under 14-h-light followed by 10-h-dark.

Extracted molecule

total RNA

Extraction protocol

Twelve µm sections of crown, lateral, and embryonic root initials tissues were put onto RNase-free PEN membrane glass slide (Carl Zeiss, 415190-9041-000). Lasser catpture microdissection (LCM) was performed using the ZEISS PALM MicroBeam system.The RNA of dissected tissues was isolated with RNeasy Plus Micro Kit (Qiagen, 74034). The DNA of dissected tissues was isolated with DNA Micro Kit (Qiagen, 56304).
For RNA-seq library construction, Smart-seq v4 Ultra Low Input RNA Kit (Takara, 091817) was used for amplifying purified mRNA. The amplified DNA was sheared to 200-500 bp range by sonication and purified with AMPure XP beads. 1 μg DNA was used for following library construction. Purified DNA was end repaired, added with adenylate in the 3’ ends, ligated with adapters, and amplified with PCR for 7-8 cycles with the reagents from TruSeq® ChIP Sample Preparation Kit (Illumina). For BS-seq library construction, Bisulfite conversion was performed by utilizing Zymo EZ Methylation Direct Kit (Zymo, D5020). Converted DNA was purified with PureLink PCR Purification Column Kit (Invitrogen, K210012) with minor modification (add M-Desulphonation Buffer to the column and hold for 15 mins before washing) as Clark et al. (2017) suggested. The converted ssDNA was then used for following BS-seq libraries construction with TruSeq® DNA Methylation Kit (Illumina).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

HiSeq X Ten

Description

all-genes-spatiotemporal-expression-matrix.txt

Data processing

For RNA-seq analysis, HISAT (v0.1.6) was used to map clean reads to rice reference genome (RGAP version 7.0). Cufflinks (v2.2.1) and Cuffdiff (v2.2.1) were used to splice transcripts and find differential expressed genes. A strict criteria (fold change>4, q value<0.01) was used to filter differentially expressed genes. For BS-seq analysis, clean reads were mapped to rice reference genome (RGAP version 7.0) by BS-seeker2 (v2.1.8). MethylKit (v3.6) was used to identify differentially methylated regions (DMRs) with win.size equal to 1000 and step.size equal to 100. To avoid bins with few cytosines, we selected bins with at least four cytosines and covered by at least ten reads. Q value less than 0.01 as well as absolute methylation difference of 0.7, 0.5, and 0.1 for CG, CHG, and CHH, respectively, were selected as Tan et al. (2016) suggested. Adjacent bins with overlapping were merged into one DMR.
Genome_build: RGAP version 7.0
Supplementary_files_format_and_content: bed files are tab-delimited text files containing gene expression profile information
Supplementary_files_format_and_content: diff.txt files are blank-delimited text files containing differentially methylated region information

Submission date

Sep 03, 2019

Last update date

Feb 01, 2020

Contact name

shaoli zhou

E-mail(s)

zhoushaoli@mail.hzau.edu.cn

Organization name

Huazhong Agricultural University

Department

National Key Laboratory of Crop Genetic Improvement