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Sample GSM2417762

Query DataSets for GSM2417762

Status

Public on Jun 13, 2017

Title

Shiranuhi etiolated seedlings, sample 3

Sample type

SRA

Source name

leaves

Organism

(Citrus unshiu x Citrus sinensis) x Citrus reticulata

Characteristics

cultivar: Shiranuhi
tissue: leaves

Growth protocol

Seeds were presoaked for 4h, and incubated in 25°C for 3d, then sowed in pots filled with vermiculite and perlite (V : V = 1 : 1). Subsequently, these pots were transferred into a growth chamber set to 25°C, 12h light/12h dark period and 50-60% relative humidity of air, and watered every two days after seedling germination.

Extracted molecule

total RNA

Extraction protocol

A total amount of 1.5 µg RNA per sample was used as input material for the RNA sample preparations. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer (5X). First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (RNase H-). Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3’ ends of DNA fragments, NEBNext Adaptor with hairpin loop structure were ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 150~200 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). Then 3 µl USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37°C for 15 min followed by 5 min at 95 °C before PCR. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. At last, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system. 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 library preparations were sequenced on an Illumina Hiseq 2000 and paired-end reads were generated.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 4000

Description

R_Y_3

Data processing

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 reads containing adapter, reads containing ploy-N and low quality reads from raw data. At the same time, Q20, Q30, GC-content and sequence duplication level of the clean data were calculated. All the downstream analyses were based on clean data with high quality. The left files (read1 files) from all libraries/samples were pooled into one big left.fq file, and right files (read2 files) into one big right.fq file. Transcriptome assembly was accomplished based on the left.fq and right.fq using Trinity with min_kmer_cov set to 2 by default and all other parameters set default. Clean data were mapped back onto the assembled transcriptome. Readcount for each gene was obtained from the mapping results.

Submission date

Dec 06, 2016

Last update date

May 15, 2019

Contact name

bo xiong

E-mail(s)

xiongbo200977@163.com

Organization name

Sichuan Agricultural University

Street address

NO.211 Huimin