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Sample GSM3703664 Query DataSets for GSM3703664
Status Public on Apr 01, 2020
Title cLP_p2mono_ko_hh_S3
Sample type SRA
Source name colonic lamina propria
Organism Mus musculus
Characteristics strain: C57BL/6 SJL
genotype: Cd45.2; Cx3cr1:GFP/+; Irf5:-/-
infection: Hh
cell type: mononuclear phagocytes FACs-isolated from the inflamed lamina propria
facs phenotype: CD45.2+ve, GFP+ve, CD11b+ve, Ly6c-hi, MHCII+ve, CD3-ve, CD19-ve, CD138-ve, NK1.1-ve, Ly6G-ve and Siglec F-ve
replicate: chimera S3
Treatment protocol Mice were infected with 1x10^8 colony forming units (cfu) Hh in 200 μL sterile PBS on days 0 and 1 by oral gavage with a 22G curved, blunted needle (Popper & Sons). Mice were injected intraperitoneally once weekly starting on day 0 with 1 mg anti-IL10R blocking antibody (clone 1B1.2) in a volume of 200 μL. Infected mice were monitored weekly for colitis symptoms. Mice were culled by Schedule 1 method three weeks after day of infection, and organs were harvested for analysis.
Growth protocol Mice were bred and maintained under SPF conditions in accredited animal facilities at the University of Oxford. All procedures were conducted according to the Operations of Animals in Scientific Procedures Act (ASPA) of 1986 and approved by the Kennedy Institute of Rheumatology Ethics Committee. Animals were housed in individually ventilated cages at a constant temperature with food and water ad libitum. Mice were free of known intestinal pathogens and negative for Helicobacter species.
Extracted molecule polyA RNA
Extraction protocol Colons and/or caeca were harvested from mice, washed in PBS/BSA and content flushed with forceps. Intestines were then opened longitudinally and washed once more before blotting to remove mucus. Gut tissue was then cut into 1 cm long pieces and placed in 50 mL centrifuge tube (Greiner) in ice cold PBS + 0.1% BSA. Colons were incubated 2 times at 200 rpm in 40 mL HBSS + 0.1% BSA + 1% Penicillin-Streptomycin (PS, Lonza) + 5mM EDTA (Sigma-Aldrich) at 37 °C for 10 min before the supernatant was aspirated. Tissue was placed in 40 mL PBS + 0.1% BSA + 1% PS for 5 min. Intestines were then incubated with 20 mL RPMI + 10% FCS +1% PS + 2.5 U/mL Collagenase VIII (Sigma-Aldrich) + 2 U/mL DNAse I (Roche), shaking at 200 rpm for 45 mins - 1 hour at 37 °C. Supernatant was filtered through a 70 μm cell strainer to which 30 mL of ice cold PBS + 0.1% BSA + 1% PS + 5 mM EDTA was added to ablate collagenase/DNase activity. Cells were washed in 30 mL PBS/BSA before filtering once more through a 40 μm cell strainer. The cells were then pelleted by centrifugation at 400 rcf for 10 minutes at 4 °C. LPLs were isolated by resuspending cells in 4 mL P80 (80% P100 (9:1 percoll:10X PBS) + 20% RPMI) percoll in a 15 mL centrifuge tube (Greiner) before overlaying 4 mL P40 (40% P100 + 60% 1X PBS) layer. Cells were spun at 2000 rcf for 20 min at room temperature, slow acceleration, no brake. Mucus and cellular debris were aspirated from the surface of the P40 layer with a Pasteur pipette and pipetted into 40 mL of ice cold PBS + 0.1% BSA. Cells were pelleted by centrifugation at 400 rcf for 10 min and resuspended in 1 mL RPMI + 10% FCS + 1% PS before counting. Cells were sorted on a FACS ARIA II cell sorter (BD) using a 100 μm nozzle. For Bulk samples, cells were sorted directly into SmartSeq II lysis buffer (Picelli et al., 2013). For 10X samples submission, cells were sorted into 1.5 mL microcentrifuge tubes coated with FCS.
For each sample 100 cells were FACs sorted into 2ul of Smart-seq2 lysis buffer and cDNA prepared according to the Smart-seq2 protocol. RNA-seq libraries were prepared using Nextera XT kits (Illumina).
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina HiSeq 4000
Description featureCounts.counts.txt
Data processing Sequence reads were aligned to the mouse genome with Hisat2 (version 2.1.0) using a “genome_trans” index built from the mm10 release of the mouse genome and Ensembl version 91 annotations (two-pass strategy to discover novel splice sites; with parameters: --dta and --score-min L,0.0,-0.2).
Mapped reads were counted using featureCounts (Subread version 1.6.3; Ensembl version 91 annotations; default parameters).
TPMs were estimated with salmon (version 0.11.3, with parameter "--gcBias") using a quasi index built from from the full set of Ensembl version 91 transcriptome annotations (parameters "--k=31 --keepDuplicates").
Genome_build: mm10
Supplementary_files_format_and_content: (1) "featureCounts.counts.txt" is a tab-delimited text file containing the table of gene-by-sample read counts computed with the featureCounts algorithm. (2) "salmon.tpms.txt" is a tab-delimited text file containing the table of gene-by-sample TPMs computed with Salmon.
Submission date Apr 02, 2019
Last update date Apr 01, 2020
Contact name Stephen Sansom
Organization name Kennedy Institute of Rheumatology
Department NDORMS
Lab Sansom
Street address Roosevelt Drive
City Oxford
State/province Oxfordshire
ZIP/Postal code OX3 7FY
Country United Kingdom
Platform ID GPL21103
Series (2)
GSE129257 IRF5 promotes intestinal inflammation by guiding monocyte differentiation towards pathogenic CD11c+ macrophage phenotype [small_bulk_rnaseq]
GSE129258 IRF5 promotes intestinal inflammation by guiding monocyte differentiation towards pathogenic CD11c+ macrophage phenotype
BioSample SAMN11322084
SRA SRX5628835

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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