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Sample GSM3769097

Query DataSets for GSM3769097

Status

Public on Mar 16, 2020

Title

osnrpd1ab_base_mRNA180209_rep1

Sample type

SRA

Source name

shoot base of seedling

Organism

Oryza sativa Japonica Group

Characteristics

tissue: shoot base
age: 4-week
treatment: none
substrate: nutrient soil

Growth protocol

Seedlings of WT and transgenic lines were grown in the green chambers at 70% humidity, under conditions with daily cycles of 12 h of light at 30 °C and 12 h of dark at 25 °C.

Extracted molecule

polyA RNA

Extraction protocol

For whole-genome bisulfite sequencing, genomic DNA was purified from the shoot base of 4-week-old seedlings using the DNeasy Plant Mini Kit (QIAGEN, 69104). For RNA sequencing, total RNA was purified from the shoot base of 4-week-old seedlings using the RNeasy Plant Mini Kit (QIAGEN, 74904). For small RNA sequencing, total RNA was purified from the shoot base of 4-week-old seedlings using TRIzol reagent (Invitrogen, 15596026). Large RNAs were selectively precipitated using 5% PEG8000 (Sigma, P5413) and 0.5 M NaCl (Sigma, S3014). Small RNAs were recovered from the supernatant by ethanol precipitation and then separated by electrophoresis using a 15% denaturing PAGE gel.
Paired-end bisulfite sequencing libraries were generated using the NEXTflex Bisulfite-Seq kit (BIOO SCIENTIFIC, NOVA-511911). Briefly, genomic DNA was sonicated to ~300 bp, end repaired and 3’ adenylated. The products were ligated with methylated adapters and then subject to size selection using Ampure XP purification beads (Beckman, A63881). Bisulfite treatment was carried out using the EZ DNA Methylation-Gold kit (ZYMO Research, D5005) according to the manufacturer’s instructions. Bisulfite-converted DNA products were amplified by PCR for 15 cycles using EX Taq DNA polymerase (Takara, RR001C). The PCR products were cleaned up using Ampure XP purification beads (Beckman, A63881). The constructed libraries were validated using the Agilent 2100 Bioanalyzer and paired-end sequenced in 150-bp on an Illumina X-Ten platform. Libraries for RNA sequencing were generated using NEXTflex RNA-Seq Kit (Bioo Scientific) according to the manufacturer’s instructions. Libraries for small RNA sequencing were generated using NEBNext Small RNA Library Prep Set for Illumina (NEB).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

HiSeq X Ten

Data processing

For whole-genome bisulfite sequencing, reads were mapped and analyzed with BRAT-BW (Harris et al., 2012). For small RNA sequencing, reads were mapped with Bowtie and analyzed by in-house PERL scripts. For RNA sequencing, reads were mapped with Tophat and differentially expressed genes were identified with DESeq2.
Genome_build: RGAP7
Supplementary_files_format_and_content: bigwig for whole-genome bisulfite sequencing; expression matrix for RNA sequencing; raw read counts for small RNA sequencing

Submission date

May 16, 2019

Last update date

Mar 16, 2020

Contact name

Yijun Qi

E-mail(s)

qiyijun@mail.tsinghua.edu.cn

Organization name

Tsinghua University

Department

School of Life Sciences