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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
Organization name Sichuan Agricultural University
Street address NO.211 Huimin
City chengdu
ZIP/Postal code 611130
Country China
Platform ID GPL22751
Series (1)
GSE90935 Transcriptome Analyses of Two Hybrid Citrus Cultivars in Seedlings Etiolation
BioSample SAMN06113112
SRA SRX2398365

Supplementary data files not provided
SRA Run SelectorHelp
Processed data are available on Series record
Raw data are available in SRA

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