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Sample GSM2450541

Query DataSets for GSM2450541

Status

Public on Jan 10, 2017

Title

Pt grazing 3

Sample type

SRA

Source name

diatom cells

Organism

Phaeodactylum tricornutum CCAP 1055/1

Characteristics

strain: CCAP 1055/1
tissue: diatom cells

Treatment protocol

To generate grazing effect, over 2000 individuals of Acartia tonsa in 25 L seawater were fed with 1×107 P. tricornutum cells per day. Acartia was cultured in two nesting buckets of with the bottom of the inner one replaced by a sieve with mesh size of 300 μm. Seawater was changed every day in the outer bucket with the feces and eggs of Acartia removed. The culture medium of Acartia was filtered with a 0.1 μm syringe filter to P. tricornutum grazing treatment groups, while the same amount of sterile-filtered seawater (for culturing Acartia) with the same ratio of the pure medium (for culturing P. tricornutum) was added to the control groups.

Growth protocol

The culture medium was prepared with sterile-filtered natural seawater from the North Sea (Minisart High-Flow 0.1μm syringe filter; Sartorius Stedim Biotech GmbH, Goettingen, Germany), and enrichment nutrient solutions (macronutrients and micronutrients) based on a modified Provasoli medium. P. tricornutum cells were grown at 18°C in a temperature-controlled room. The light intensity was constant at 100 μmol photons·m-2·s-1 at a light:dark cycle of 12:12 h with 2 hours sunrise and 2 hours sunset phase simulations via irradiance profiles as typical at this latitude.

Extracted molecule

total RNA

Extraction protocol

The total RNA was extracted with RNeasy® Plant Mini Kit (QIAGEN, Hilden, Germany).5µg total RNA was treated with Ribo-Zero™Magnetic Kit (Plant Leaf)) （Epicentre）to depleterRNA.
The retrieved RNA is fragmented by adding First Strand Master Mix（Invitrogen）. First-strand cDNA is generated by First Strand Master Mix and Super Script II reverse transcription (Invitrogen) (Reaction condition：25℃for 10 min；42℃for 50 min；70℃for 15 min).Purify the product(Agencourt RNAClean XP Beads，AGENCOURT), then add Second Strand Master Mix and dATP,dGTP, dCTP, dUTP mix to synthesize the second-strand cDNA (16℃for 1h).Then the purified cDNA combine with End Repair Mix, incubate at 30℃for 30min. After purification with beads, add A-Tailing Mix , incubate at 37℃for 30 min. Combine the Adenylate 3'Ends DNA, Index Adapter and Ligation Mix, incubate at 30℃for 10 min. After that, add the Uracil-N-Glycosylase enzyme into the purified ligation product, incubate at 37℃for 10min. Several rounds of PCR amplification with PCR Primer Cocktail and PCR Master Mix are performed to enrich the cDNA fragments. Then thePCR products are purified with Ampure XP Beads.The final library is quantitated in two ways:determine the average molecule length using the Agilent 2100 bioanalyzer instrument (Agilent DNA 1000 Reagents), and quantify the library by real-time quantitative PCR (QPCR) (TaqMan Probe).The Qualified libraries will amplify on cBot to generate the cluster on the flowcell (TruSeq PE Cluster Kit V3–cBot–HS，Illumina). And the amplified flowcell will be sequenced pairend on the HiSeq 2000 System (TruSeq SBS KIT-HS V3，Illumina), read length 90 are the most common sequencing strategy.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2500

Data processing

We use BWA to map clean reads to genome reference and use Bowtie to gene reference.
Bowtie 2 version 2.2.5 ：parameter：-q --phred64 --sensitive --dpad 0 --gbar 99999999 --mp 1,1 --np 1 --score-min L,0,-0.1 -I 1 -X 1000 --no-mixed --no-discordant -p 16 -k 200
BWA (alignment via Burrows-Wheeler transformation) Version: 0.7.10-r789 ：parameter： -o 1 -e 63 -i 90 -L -k 2 -l 31 -t 4 -q 10
Genes and isoforms expression level are quantified by a software package: RSEM v1.2.12 rsem-calculate-expression --paired-end –bam Phaeodactylum-tricornutum-control-1.bam -p 8 rsem-reference/refMrna.fa outdir/ Phaeodactylum-tricornutum-control-1
transcript are blasted to the Gene IDs are screened on KEGG, gene ontology, and GenBank to find the Kegg orthology,GO terms,and blast nr the software is blastall,version 2.2.23,the parameters are blastall -d KEGG/59.3/plant.fa -i GCF_000150955.2_ASM15095v2_rna.fna -o GCF_000150955.2_ASM15095v2_rna.fna.ko -blast blastx -e 1e-05 -m 8
Genome_build: Genome reference : GCF_000150955.2_ASM15095v2_genomic.fna Gene reference:GCF_000150955.2_ASM15095v2_rna.fna
Supplementary_files_format_and_content: tab-delimited text files include FPKM values, Kegg orthology,GO terms,and blast nr for each Sample

Submission date

Jan 09, 2017

Last update date

May 15, 2019

Contact name

Si Li

E-mail(s)

jackeikee@aliyun.com

Organization name

Hebei University of Technology

Street address

Guangrong Dao 8