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Sample GSM777474

Query DataSets for GSM777474

Status

Public on Dec 30, 2013

Title

microRNAs from MDV-infected tumorous spleen

Sample type

SRA

Source name

MDV-infected tumorous spleen

Organism

Gallus gallus

Characteristics

breed: Specific pathogen free White Leghorn (BWEL)
tissue-type: Spleen
development stage: 46 days post infection

Treatment protocol

One hundred chickens from treatment group were infected intraperitoneally with 2000 PFU of the MDV GA strain (passage 9) and fifty chickens from controls were injected with the same dosage of diluent (0.2 mL) as controls at one day old. The two groups were kept in separate isolators in different room. The whole trial period lasted to 56 days postinfection (d. p. i.). During this phase, we observed the chicken' clinical signs 2-3 times daily and chose the dying ones to euthanize. Spleen, liver, and lymphocytes isolated from peripheral blood were collected. Meanwhile, chickens from age-matched control were disposed in the same way. All tissues and lymphocytes were immediately stored in RNAfixer at 4â overnight and transferred to -80â until RNA isolation. Finally, we chose four samples, including uninfected spleen and lymphocytes from control and lymphoma in liver and tumorous spleen from MDV-infected chickens to perform high-throughtput solexa deep sequencing.

Growth protocol

One hundred and fifty day-old chickens from White Leghorn specific pathogen free line (BWEL) were randomly divided into two groups. One hundred chickens were treatment group and the other 50 were controls. The two groups were kept in seeprate cages with the same filtered-air, positive-pressure condtion, and provided with water and commercial feed ad libitum.

Extracted molecule

total RNA

Extraction protocol

Total RNA of four samples was extracted using mirVana miRNA Isolation Kit (Ambion, Austin, USA) according to the manufacturer's protocol. Briefly, 10 Î¼g of RNA sample was size fractionated with YM-100 column (Millipore). The larger RNA fraction (>100 KDa) was removed. The smaller RNA fraction (<100KDa) was further fractioned on a 15% tris-borate-EDTA (TBE) urea polyacrylamide gel and 15-50nt fraction was obtained. Subsequently, 5' and 3' RNA adapters were ligated to the small RNA by T4 RNA ligase (Promega), followed by reverse transcription and PCR products purification. Purified products were quantified on the TBS-380 mini-fluorometer (Turner Biosystems) using Picogreen? dsDNA quantitation reagent (Invitrogen) and diluted to 10 nM and transferred for sequencing on the Illumina/Solexa G1 sequence (Illumina, San Diego, USA ).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

size fractionation

Instrument model

Illumina Genome Analyzer

Description

microRNAs from MDV-infected tumorous spleen

Data processing

The raw sequenced sequences (sequ-seqs) were subjected to Illumina pipeline filter (Solexa 0.3). Each of the pipeline filter treated data set was further processed with ACGT101-miR, including classify unique sequence family, adapter (ADT) dimmer (5' ADT, 3' ADT & 5' ADT and 3'ADT hooked together without insertion) filter, junk (80% of A, or C, or G, or T, or 3N; 10 repeat of dimmer, 6 repeat of trimmer, or 5 repeat of tetramer; 7 consecutive A, 8 consecutive C, 6 consecutive G or 7 consecutive T; contained only A and C or only G and T) filter, length filter (<16 nt or > 26 nt were removed), simple sequence ( 80% of A, or C, or G, or T, or 3N) filter, low copy (< 3) filter, mRNA, RFam & repbase filter (unique seqs were blasted against mRNA, RFam & repbase. If a unique sequence hits any of the mRNA, or RFam & repbase with 1 error allowed, it was filtered out). After a series of filtering, the remaining sequences (we called mappable sequences) were blasted to miRBase 16.0 and genome (chicken (WUGSC 2.1/galGal3, May 2006) and MDV(AF147806.1)) to identify known and novel miRNAs. Length variation at both 3' and 5 ends and one mismatch inside of the sequence were allowed in alignment.

Submission date

Aug 12, 2011

Last update date

May 15, 2019

Contact name

Ling Lian

E-mail(s)

lianlinglara@126.com

Organization name

China Agricultural University

Street address

Room 129, Animal Science Department, 2nd Yuanmingyuan West Road, Haidian District