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Sample GSM5658115

Query DataSets for GSM5658115

Status

Public on Oct 21, 2024

Title

R308_7

Sample type

SRA

Source name

filling panicles

Organism

Oryza sativa

Characteristics

tissue: filling panicles
line: R308 (Parent)

Treatment protocol

Three replicates were setted.

Growth protocol

All the tested lines were routinely planted in the experimental field of Dafeng base.

Extracted molecule

total RNA

Extraction protocol

RNA library for lncRNA-seq was prepared as rRNA depletion and stranded method. Briefly, the ribosomal RNA was depleted from total RNA using the rRNA Removal Kit following manufacturer’s instruction. RNA was then fragmented into 250~300 bp fragments, and first strand cDNA was reversetranscribed using fragmented RNA and dNTPs (dATP, dTTP, dCTP and dGTP). RNA was degraded using RNase H, and second strand cDNA was synthesised using DNA polymerase I and dNTPs (dATP, dUTP, dCTP and dGTP). Remaining overhangs of double-strand cDNA were converted into blunt ends via exonuclease/ polymerase activities. After adenylation of 3’ ends of DNA fragments, equencing adaptors were ligated to the cDNA. In order to select cDNA fragments of preferentially 250~300 bp in length, the library fragments were purified with AMPure XP system. Uridine digestion was performed using Uracil-N-Glycosylase, which was followed by the cDNA amplification using PCR
RNA librarys were prefared for secquencing using standard Illumina protocls

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 4000

Data processing

The clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS (Illumia) according to the manufacturer’s instructions. After cluster generation, the libraries were sequenced on an Illumina Hiseq 4000 platform and 150 bp paired-end reads were generated.
Raw data (raw reads) of FASTQ format were firstly processed through in-house perl scripts. In this step, clean data (clean reads) were obtained by removing following reads: (1) reads with 5’ adapter; (2) reads without 3’ adapter or insert sequence; (3) reads with more than 10% N; (4) reads with more than 50% nucleotides with Qphred<=20; (5) reads with ploy A/T/G/C. Adapter trimming for the removal of adapter sequences from the 3’ ends of reads was also performed. At the same time, Q20, Q30 and GC content of the clean data were calculated. All the downstream analyses were based on the clean data with high quality. Clean reads for each sample were first mapped to a reference genome with the software HISAT2.
Reads Per Kilobase of exon per Megabase of library size (RPKM) were calculated using a protocol from Chepelev et al., Nucleic Acids Research, 2009. In short, exons from all isoforms of a gene were merged to create one meta-transcript. The number of reads falling in the exons of this meta-transcript were counted and normalized by the size of the meta-transcript and by the size of the library.
Genome_build: R498_mbkbase
Supplementary_files_format_and_content: Matrix table with fpkm for every gene and every sample

Submission date

Oct 26, 2021

Last update date

Oct 21, 2024

Contact name

Ma Ce

E-mail(s)

290905629@qq.com

Phone

15222602669

Organization name

Nanjing