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| Status |
Public on Nov 26, 2020 |
| Title |
input_memory_H3K27ac_4 |
| Sample type |
SRA |
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| Source name |
flow sorted bone marrow inflammatory monocytes
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| Organism |
Mus musculus |
| Characteristics |
infection: 30 days after drug cure of chronic mosquito transmitted P. chabaudi AJ
chip antibody: not immunoprecipitated: input
tag: C57Bl/6J
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| Treatment protocol |
30 days after chloroquine treatment of once-P. chabaudi AJ malaria infected mice and uninfected age-matched controls, bone marrow cells from both femurs and tibias (pooled from two mice) were stained with antibodies and gently fixed in PBS with 1 % paraformaldehyde (Alpha Aesar) and 10 % FBS for exactly 10 minutes at room temperature. Fixation was quenched with 125 mM Glycine (Sigma). 50,000 bone marrow inflammatory monocytes (Lineage- Ly6G- cKit- CD135- CD11b+ Ly6C+) were then flow-sorted into polypropylene tubes with IMDM supplemented with 5 % FBS and 8 mM L-Glutamine. We flow-sorted multiple replicates from one biological sample, which were then chromatin-immunoprecipitated using three different antibodies. Sorted monocytes were pelleted by centrifugation and washed in HBSS (Gibco) with protease inhibitors (complete ULTRA Tablets Protease Inhibitor Cocktail, Roche) and 5 mM sodium butyrate (Alpha Aesar). Cell pellets were stored at - 80 ̊C.
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| Growth protocol |
C57Bl/6J mice were bred and housed in individually ventilated cages under specific pathogen free conditions (22°C, 50% humidity) at the University of Edinburgh, UK. Since we have previously shown that mosquito transmission resets expression of the large sub-telomeric multi-gene families that control malaria parasite virulence, and in turn shapes the host immune response during the pathogenic blood-stage of infection (Spence, P.J., et al., Vector transmission regulates immune control of Plasmodium virulence. Nature, 2013. 498(7453): p. 228-31), we transmitted Plasmodium chabaudi chabaudi AJ 96AJ15 (obtained from the European Malaria Reagent Repository (malariaresearch.eu)) through Anopheles stephensi mosquitoes (strain SD500, reared in house at the University of Edinburgh) according to our published protocol: Spence, P.J., et al., Mosquito transmission of the rodent malaria parasite Plasmodium chabaudi. Malar J, 2012. 11: p. 407. Mice were injected intravenously with 200 P. chabaudi AJ sporozoites, which, following 52 hours development in the liver, establish a severe acute blood-stage infection (hyperparasitaemia, severe anaemia, hypothermia and prostration) lasting approximately 2 weeks. Thereafter > 50 % of mice remain chronically infected (verified after 40 days by quantitative PCR). Chronic P. chabaudi AJ infection was then drug treated using 100 mg/kg chloroquine diphosphate salt (Sigma, dissolved in water) administered by oral gavage daily for 10 days. Memory responses of once-malaria infected mice were assessed 30 days after the start of chloroquine treatment and compared to uninfected age-matched control mice that received an identical chloroquine regimen.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Chromatin was immunoprecipitated using the True MicroChIP kit (Diagenode) with a modified protocol: cells were lysed before chromatin shearing using a Bioruptor Pico Sonicator (Diagenode; 5 cycles: 30 seconds ON - 30 seconds OFF; fragments 100 - 300 bp). 10 % of sheared chromatin was kept as input control and 90 % was immunoprecipitated using antibodies against either H3K27ac, H3K4me1 or H3K9me (all Diagenode) over night at 4 ̊C. The next morning Protein A coated beads (Diagenode) were added and, following a 6 hour incubation at 4 ̊C, unbound chromatin fragments were removed by thorough washing. ChIP and input DNA was then decrosslinked from proteins and purified using MicroChip DiaPure columns (Diagenode).
Sequencing libraries were prepared using the MicroPlex v2 Library preparation kit (Diagenode) according to manufacturers instructions. Library amplification was monitored in real time using a Lightcycler 480 (Roche) to determine optimal cycle number. Amplified libraries were quantified using Qubit (dsDNA HS assay) and fragment quality and size distribution assessed by Bioanalyser (DNA HS Kit). Following equimolar pooling, libraries were purified using AMPure XP beads and quantity and quality of the library pool confirmed. Pooled libraries were sequenced by Edinburg Genomics, University of Edinburgh (UK) on HiSeq 4000 (75 bp paired end reads) or NovaSeq S1 (100 bp paired end reads, both Illumina).
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
H3K27ac_MEMORY_INPUT_AJ
H3K27ac_MEMORY_INPUT_AJ.tagdir.tar.gz
H3K27ac_MEMORY_INPUT_AJ.bedgraph
H3K27ac_MEMORY_INPUT_AJ.tdf.gz
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| Data processing |
Raw sequence data were assessed for quality and content using FastQC
Sequences were aligned to the Mus musculus genome (mm10) using bowtie2 with parameters --very-sensitive -p 30 --no-discordant --no-unal . The bowtie2 alignment outputs were stored as sorted, indexed bam files generated using samtools.
Samples were pooled as detailed and converted into tag directories using Homer software (http://homer.ucsd.edu/homer/; v4.10)
Alignments were converted to bedgraph format using Homer script makeUCSCfile.
Wig format outputs were converted to TDF format for viewing in IGV using igvtools v2.3.93 (parameters: toTDF -z 7 -f p98)
Genome_build: mm10
Supplementary_files_format_and_content: Three types of processed file are provided for each sample pool. Firstly, a tarred gzip archive of the Homer derived tag counts. Secondly, the tags were converted into bedgraph format, prior to conversion to wiggle format. Thirdly, the wiggle format files were coverted to a comcpressed TDF format more suitable for IGV viewing.
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| Submission date |
May 13, 2020 |
| Last update date |
Nov 26, 2020 |
| Contact name |
Alasdair Ivens |
| E-mail(s) |
al.ivens@ed.ac.uk
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| Phone |
44 131 6513605
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| Organization name |
Centre for Immunity, Infection and Evolution
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| Street address |
Kings Buildings
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| City |
Edinburgh |
| ZIP/Postal code |
EH9 3FL |
| Country |
United Kingdom |
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| Platform ID |
GPL24247 |
| Series (2) |
| GSE150478 |
Bone marrow monocytes from once-malaria infected mice have no epigenetic memory of the infection. |
| GSE150479 |
Inducible mechanisms of disease tolerance provide an alternative strategy of acquired immunity to malaria. |
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| Relations |
| BioSample |
SAMN14911552 |
| SRA |
SRX8336473 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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