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Sample GSM3265182

Query DataSets for GSM3265182

Status

Public on Mar 20, 2019

Title

KO90min_2

Sample type

SRA

Source name

Splenocytes

Organism

Mus musculus

Characteristics

strain: C57BL/6
tissue: spleen
age: 8 weeks
genotype: GdX-/-

Treatment protocol

The wilde type and GdX-/Y mice were injected with LPS (20 mg/kg body weight, i.p.), and the spleen was collected at 90 min or 6 hrs after injection.

Extracted molecule

total RNA

Extraction protocol

The spleen was collected and smashed on ice. Total RNA samples were harvested using Trizol reagent. We use Agilent 2100 Bio analyzer (Agilent RNA 6000 Nano Kit) to do the total RNA sample QC: RNA concentration, RIN value,28S/18S and the fragment length distribution.
1. mRNA enrichment: Oligo dT Selection or rRNA depletion. 2. RNA fragment and reverse transcription: Fragment the RNA and reverse transcription to double-strand cDNA (dscDNA) by N6 random primer. 3. End repair, add A tailing and adaptor ligation:The synthesized cDNA was subjected to end-repair and then was 3’ adenylated. Adaptors were ligated to the ends of these 3’ adenylated cDNA fragments. 4. PCR amplification:The ligation products were purified and many rounds of PCR amplification were performed to enrich the purified cDNA template using PCR primer. 5. Denature and cyclization:Denature the PCR product by heat and the single strand DNA is cyclized by splint oligo and DNA ligase. 6.Sequencing on BGISEQ- 500 platform.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

BGISEQ-500

Data processing

The software for base calling is BaseCallV1.6.0.19379
We use internal software SOAPnuke to filter reads, followed as: (1) Remove reads with adaptors;(2) Remove reads in which unknown bases(N) are more than 10%; (3) Remove low quality reads (we define the low quality read as the percentage of base which quality is lesser than 15 is greater than 50% in a read).After filtering, the remaining reads are called "Clean Reads" and stored in FASTQ format.
We use HISAT (Hierarchical Indexing for Spliced Alignment of Transcripts) to do the mapping step. The software for base calling is HISAT2 v2.0.4, -p8--phred64--sensitive-I 1-X 10000.
We filter the low quality reads (More than 20% of the bases qualities are lower than 10), reads with adaptors and reads with unknown bases (N bases more than 5%) to get the clean reads.Then we assembled those clean reads into Unigenes, followed with Unigene functional annotation, SSR detection and calculate the Unigene expression levels and SNPs of each sample. Finally, we identify DEGs (differential expressed genes) between samples and do clustering analysis and functional annotations.
We mapped clean reads to reference using Bowtie2, and then calculate gene expression level with RSEM. RSEM is a software package for estimating gene and isoform expression levels from RNA-Seq data. Then, we calculate pearson correlation between all samples using cor, perform hierarchical clustering between all samples using hclust, perform PCA analysis with all samples using princomp, and draw the diagrams with ggplot2 with fuctions of R. Software information: Bowtie2 : Version: v2.2.5 Parameters: -q --phred64 --sensitive --dpad 0 --gbar 99999999 --mp 1,1 --np 1 --score-min L,0,-0.1 -p 16 -k 200
Genome_build: NCBI_GRCm38.p5
Supplementary_files_format_and_content: The processed data files include FPKM values for all the samples.

Submission date

Jul 11, 2018

Last update date

Mar 20, 2019

Contact name

Yifan Zhou

E-mail(s)

[email protected]

Organization name

Tsinghua University

Department

School of Medicine