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Sample GSM4600741

Query DataSets for GSM4600741

Status

Public on Jun 09, 2020

Title

mutant pp1r3-2 2

Sample type

SRA

Source name

leaf

Organism

Arabidopsis thaliana

Characteristics

ecotype: Columbia-0
tissue: leaf
age: 21 day after germination
genotype/variation: mutant pp1r3-2

Treatment protocol

Normal growth conditions, wild type (Col) as a control

Growth protocol

Arabidopsis (Columbia, Col) was used in the present study. And the plants used for RNA-seq were grown in a greenhouse under a 16-h light/8-h dark photoperiod at 23℃.

Extracted molecule

total RNA

Extraction protocol

Novogene uses standard extraction methods to extract RNA from tissues or cells, and then strictly controls the RNA samples. RNA quantification and qualification ①RNA degradation and contamination was monitored on 1% agarose gels. ② RNA purity was checked using the NanoPhotometer ® spectrophotometer (IMPLEN, CA, USA) . ③ RNA integrity was assessed using the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies, CA, USA).
A total amount of 1 µg RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer’s recommendations and index codes were added to attribute sequences to each sample. 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 250~300 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

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

Description

processed data_gene_count.xls [r3_2]

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 library preparations were sequenced on an Illumina Novaseq 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 reads containing adapter, reads containing ploy-N and low quality reads from raw data. At the same time, Q20, Q30 and GC content the clean data were calculated. All the downstream analyses were based on the clean data with high quality
Reference genome and gene model annotation files were downloaded from genome website directly. Index of the reference genome was built using Hisat2 v2.0.5 and paired-end clean reads were aligned to the reference genome using Hisat2 v2.0.5. We selected Hisat2 as the mapping tool for that Hisat2 can generate a database of splice junctions based on the gene model annotation file and thus a better mapping result than other non-splice mapping tools.
Genome_build: Arabidopsis_thaliana_Ensemble_44
Supplementary_files_format_and_content: Excel table contains the gene readcount of each gene in each sample and the basic information of the gene.

Submission date

Jun 08, 2020

Last update date

Jun 09, 2020

Contact name

Jing Zhang

E-mail(s)

zhangjing2011201@126.com

Phone

+8613519654901

Organization name

Fujian Agriculture and Forestry University