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Sample GSM944947

Query DataSets for GSM944947

Status

Public on Aug 01, 2013

Title

Sheep 3 Pre

Sample type

SRA

Source name

Afferent lymph myeloid cells, pre-challenge

Organism

Ovis aries

Characteristics

treatment: vaccination with liposomes+poly (I:C)+ diptheria toxoid
time post-vaccination: pre-challenge
cell type: afferent lymph myeloid cells
sample id: 3b

Treatment protocol

Lymphatic collections were obtained prior to vaccination and at 4-6 hours, 26-28 hours, 51-53 hours and 76-78 hours post vaccination. Experimental lymph samples were collected in sterile 50-ml tubes containing 0.05 IU of heparin (Pfizer, Parkville, VIC, Australia) and 20 ml of 100x cell culture penicillin-streptomycin (Invitrogen, Carlsbad, CA, USA). Only cannulation sites with continuous lymph flow over the observation period were used for data analysis. Lymphocytes were depleted using antibody coupled magnetic depletion. Lymph cells were collected for 2 hours, red cell lysis was performed and the pellet was resuspended in 1 ml of depletion mix (anti-CD45R clone 20.96 (neat hybridoma supernatant), anti-CD4 clone 44.38 (1/100 hybridoma supernatant) and anti γδ TCR clone 86D (1/25 hybridoma supernatant). The mix was incubated on ice for 15 min, and then 9ml of FACS wash was added and the cells centrifuged. The pellet was washed twice with 10 ml of FACS wash and then resuspended in FACS wash at 2x10^7 cells/ml. The magnetic beads coupled with anti-mouse Ig (BioMag, Polysciences Inc., Warrington, PA) were washed 3 times with 5 ml of FACS wash and added to the cells for depletion at 5 beads per cell. The mixture of beads and cells were gently rotated for 20 minutes at 4 oC before the tubes were placed beside a neodynium magnet until the solution cleared. The process was repeated on the supernatant to ensure all beads were removed. The main cell pellet was resuspended in 350 ul of RLT (Qiagen) and total RNA was extracted using an RNeasy Micro Plus Kit (Qiagen, Valencia, USA) according to the manufacturer's instructions. RNA quantity was measured using a Nanodrop (Nanodrop Technologies, Wilmington, USA) and RNA quality was assessed using an Agilent Bioanalyzer (Agilent Technologies, Foster City, USA).

Growth protocol

This study employed an ovine model of pseudoafferent lymphatic cannulation (see de Veer et al. 2010, Vaccine) to characterise the innate immune response within the afferent lymph to vaccination with liposomes+poly (I:C)+ diptheria toxoid. The sheep used in this study had both their pre-femoral lymph nodes removed at 1 year of age. Approximately 1 year after the lymph node removal, a second surgery was performed to insert a 0.96_0.58mm heparin-coated polyvinyl chloride cannula into the pseudoafferent (previous efferent) lymphatic duct of both sides. At least seven days were allowed for healing to occur after surgery before vaccinations were administered and experimental lymph samples were collected. Handling of animals and experimental procedures were all approved by the Monash University Animal Ethics Committee in accordance with the relevant licensing agreement. Vaccinations were injected subcutaneously in the area drained by the prefemoral lymph node. Afferent lymphatic samples were collected prior to vaccination as a control (PRE) and at 4-6, 26-28, 51-53 and 76-78 hours post vaccination with liposomes+poly (I:C)+ diptheria toxoid.

Extracted molecule

total RNA

Extraction protocol

Total RNA was extracted from cells preserved in buffer RLT using a Qiagen RNeasy Mini Kit. Only two samples yielded sufficient RNA for sequencing at the 4-6 and 26-28hrs time points. Three samples were used for all other time points. mRNA sequencing libraries were then prepared using an Illumina mRNA-seq-8 sample preparation kit. Briefly, poly-A mRNA was purified from total RNA using poly-T-oligo attached magnetic beads. The RNA was then fragmented into small pieces using divalent cations under elevated temperature. First strand cDNA synthesis was then performed using reverse transcriptase and random primers and was followed by second strand synthesis with DNA polymerase I and RNase H. End repair processing, polyadenylation and ligation of adaptors was then performed. The product was then purified and enriched by PCR.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2000

Data processing

As limited annotation data is available for ovine genes, the reads were aligned to mRNA sequences from the Bos taurus genome (NCBI build 5, version 2) as validated previously (Jager, 2011 BMC Genomics (PMID 21435219)). Reads were aligned to the bovine genome using the SHRiMP read aligner.
Read alignment counts per gene were analysed using edgeR (Robinson et al., 2010). Genes with less than 10 alignments in total were discarded. EdgeR uses a Generalized Linear Model with a log link function and a negative binomial noise distribution. The dispersion was estimated using EdgeR's trend mode, that is, dispersion was taken to be a smoothly varying function of the total count for each gene. A likelihood ratio test was performed to identify genes whose expression changed significantly over time.
Genome_build: Bos taurus NCBI build 5, version 2.
Supplementary_files_format_and_content: Data from all samples is combined into a single text file, which is linked to the Series record as a supplementary file. Expression is displayed as log2 average fold-change relative to matched pre-vaccination controls. Feature = Entrez Gene ID; log2 concentration = Log2 of the proportion of alignments; log2 timeXXX = Log2 fold-change between PRE and the specified time; p = p-value from likelihood ratio test; FDR = False Discovery Rate (adjusted p-value); Gene ID= Feature; Gene= Gene symbol; Product= Gene name or description; Note= Additional gene information; [Sample ID] = Raw count of alignments to gene per sample; RPKM [Sample ID] = Reads Per Kilobase of exonic sequence per Million mapped reads; Ambiguous alignment = Number of times there was a read that aligned equally well to other genes.

Submission date

Jun 06, 2012

Last update date

May 15, 2019

Contact name

Melissa Burke

E-mail(s)

[email protected]

Organization name

Monash University

Department

Physiology