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Status |
Public on Nov 11, 2014 |
Title |
Ago1-associated miRNA Seq |
Sample type |
SRA |
|
|
Source name |
28d worm
|
Organism |
Schistosoma japonicum |
Characteristics |
geographic origin of sample: Anhui province of China host: New Zealand white rabbit gender: male and female antibody: anti-SjAgo1; non-commercial antibody
|
Treatment protocol |
The worms were collected at desired time point from the mesenteric veins and liver of infected rabbit, respectively.
|
Growth protocol |
In order to collect S.japonicum worms(28d)), New Zealand white rabbits were infected percutaneously with cercariae of S. japonicum (400worms/rabbit) which shed from naturally infected snails.
|
Extracted molecule |
total RNA |
Extraction protocol |
We isolated and identified the small RNAs associated with the S. japonicum Argonaute protein, SjAgo1, by HITS-CLIP Libraries were prepared according to Illumina's instructions accompanying the DNA Sample Kit (Part# 0801-0303). Briefly, DNA was end-repaired using a combination of T4 DNA polymerase, E. coli DNA Pol I large fragment (Klenow polymerase) and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow fragment (32 to 52 exo minus) and dATP to yield a protruding 3- 'A' base for ligation of Illumina's adapters which have a single 'T' base overhang at the 3’ end. After adapter ligation DNA was PCR amplified with Illumina primers for 15 cycles and library fragments of ~250 bp (insert plus adaptor and PCR primer sequences) were band isolated from an agarose gel. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the Genome Analyzer following the manufacturer's protocols.
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|
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Library strategy |
RIP-Seq |
Library source |
transcriptomic |
Library selection |
other |
Instrument model |
Illumina Genome Analyzer |
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|
Description |
Protocol: HITS-CLIP
|
Data processing |
Illumina Casava1.7 software used for basecalling. The sequence tags from Soxla sequencing will go through the data cleaning analysis to get credible clean tags. We will get rid of some contaminant reads from the fq file and get the final clean reads. The data is processed by the following steps:1) Getting rid of low quality reads (The criteria for this is listed in the explanation of meaning of each row in the result table)2) Getting rid of reads with 5' primer contaminants3) Getting rid of reads without 3' primer4) Getting rid of reads without the insert tag5) Getting rid of reads with poly A6) Getting rid of reads shorter than 18nt7) Summarize the length distribution of the clean reads. Tags map to genome by SOAP or bowtie to analyze their expression and distribution on the genome. Then the standard analysis will annotate the clean tags into different categories and take those which can not be annotated to any category to predict the novel miRNA and seed edit of potential known miRNA.After getting miRNAresult,target prediction target genes will be analysed. Tags align to miRBase database using blast or bowtie,indentify known miRNA.Known miRNAs also use to subsequent analysis. We developed a prediction software mireap(animal/plant) or mirdeep(animal) to predict novel Differential expression analysis of miRNA with Known miRNA and Novel miRNA. ExpDiff method is used which compare the known or novel miRNA expression between two samples to find out the differentially expressed miRNA with logR>1 and pvalue <0.05. The target predition is used RNA hybrid software Genome_build: ASM15177v1 Supplementary_files_format_and_content: miRNA-Known.fa The miRNAs whose sequences are similar with miRNAs deposited at miRBase,fasta format. mir-novel.fa The mature novel miRNA data,fasta format. tag_CLIP1_known.txt The Known miRNAs and its predicted targets by our sequence program, txt format.
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|
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Submission date |
Nov 10, 2014 |
Last update date |
May 15, 2019 |
Contact name |
Zhao Jing |
E-mail(s) |
[email protected]
|
Organization name |
Tongji university
|
Street address |
1239 Siping Road
|
City |
Shanghai |
ZIP/Postal code |
200092 |
Country |
China |
|
|
Platform ID |
GPL11650 |
Series (1) |
GSE63145 |
HITS–CLIP reveals Argonaute 1-associated MicroRNAs and Target Sites in Schistosoma japonicum |
|
Relations |
BioSample |
SAMN03174521 |
SRA |
SRX756969 |