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| Status |
Public on Jun 11, 2020 |
| Title |
pbmc_mono_wt_ss |
| Sample type |
SRA |
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| Source name |
preipheral blood
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| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6 SJL
genotype: Cd45.1
facs phenotype: CD45+CD11b+ SiglecF-Ly6G-CX3CR1+Dump-
replicate: pool of cells from n=5 chimeras
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| 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.
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
(1) Isolation of lamina propria leukocytes: 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. Colonic lamina propria leukocytes (cLPLs) 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 3000 rpm 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. (2) Isolation of blood leukocytes: Blood was harvested by either tail vein bleed or cardiac puncture. Mice were culled by Schedule 1 method in accordance with the project licence. Prior to cardiac puncture a 1 mL syringe was coated with PBS + 2 mM EDTA. Cardiac puncture was performed with a 27G needle. Tail vein bleeds were performed using a #24 blade scalpel (Swann-Morton Ltd.) Collected blood was placed in 1 mL of sterile 2 mM EDTA/PBS solution in a 15 mL centrifuge tube (Greiner). Tubes were topped up with ice-cold PBS + 0.1% BSA. Cells were pelleted by centrifugation at 400 rcf for 10 mins at 4 °C and the supernatant discarded. Erythrocytes were then lysed using 10-20X the blood sample volume of ACK lysis buffer (Gibco) for 3 mins. Tubes were then topped up to 15 mL with ice cold PBS + 0.1% BSA to quench the lysis buffer. Cells were washed in PBS/BSA, resuspended in PBS + 0.1% BSA to the desired cell concentration, and stored at 4 °C until required.
Libraries were generated using 10x Genomics Single Cell 3’ Solution (version 3) kit and subjected to Illumina sequencing (NovaSeq 6000).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
Data analysis was performed using Python3 pipelines (https://github.com/sansomlab/tenx) written using CGAT-core. Read mapping, quantitation and aggregation of sample count matrices was performed with the 10x Genomics Cell Ranger pipeline (version 3.1.0). For the "cellranger count" step, a custom reference was built using Ensembl annotations (version 91) that included genes with protein coding, lincRNA, macro_lincRNA, immune (IG_*, TR_*), antisense_RNA, and miRNA Ensembl (version 91) biotypes.
Within each dataset, Irf5-/- and wildtype samples were aggregated. No normalisation was applied during the aggregation step. Cells with barcodes common to more than one sample from the same sequencing batch were removed from the analysis to avoid issues associated with index hopping. For each of the experiments, the aggregated count matrices were randomly down-sampled in order to normalise the median number of UMIs per-cell between the Irf5-/- and wildtype samples ("downsampleMatrix" function from the DropletUtils R package) and genes detected in less than 3 cells removed.
For the steady state colon dataset, cells with < 1k genes, > 25k UMIs, > 7.5% mitochondrial UMIs, identified as contaminating B, T or stromal cells (n=75), or with high expression of interferon-induced genes (n=34) were removed. For the steady state PBMC dataset, cells with < 500 genes, > 20k UMIs, > 5% mitochondrial UMIs, with high expression of haemoglobin genes (n=8) or identified as contaminating T cells (n=694) were removed. For each of the datasets either the WT or Irf5-/-cells were randomly down sampled to retain an equal number of cells per genotype.
Genome_build: mm10
Supplementary_files_format_and_content: The "matrix.mtx.gz" MEX file contains the aggregated count matrix (cells in columns, genes in rows). The "barcodes.tsv.gz" TSV file contains the cell barcodes (tissue and genotype are indicated in the cell barcode with the suffixes "-cLP_wt", "-cLP_ko", "-pbmc_wt", "-pbmc_ko"). The "genes.tsv.gz" file contains the Ensembl gene identifiers and names. These files can be read with the "Read10X" function from the Seurat R package.
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| Submission date |
Jun 10, 2020 |
| Last update date |
Jun 11, 2020 |
| Contact name |
Stephen Sansom |
| E-mail(s) |
stephen.sansom@kennedy.ox.ac.uk
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| Organization name |
Kennedy Institute of Rheumatology
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| Department |
NDORMS
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| Lab |
Sansom
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| Street address |
Roosevelt Drive
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| City |
Oxford |
| State/province |
Oxfordshire |
| ZIP/Postal code |
OX3 7FY |
| Country |
United Kingdom |
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| Platform ID |
GPL24247 |
| Series (2) |
| GSE129258 |
IRF5 promotes intestinal inflammation by guiding monocyte differentiation towards pathogenic CD11c+ macrophage phenotype |
| GSE152198 |
IRF5 promotes intestinal inflammation by guiding monocyte differentiation towards pathogenic CD11c+ macrophage phenotype [scrnaseq_steady_state] |
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| Relations |
| BioSample |
SAMN15198490 |
| SRA |
SRX8518686 |
| 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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