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GEO help: Mouse over screen elements for information. |
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Status |
Public on Dec 16, 2021 |
Title |
SMK-4m-1 |
Sample type |
SRA |
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Source name |
Lung tissue
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Organism |
Mus musculus |
Characteristics |
tissue: Lung tissue air exposure: cigarette smoke time point: 4 months number of cells: 2437
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Treatment protocol |
Cigarette smoke (CS) was generated from 3R4F Research Cigarettes, with the filters removed. Mice were whole body exposed to active 100% mainstream CS of 500 mg/m3 total particulate matter for 50 min twice per day for either 2 or 4 months in a manner mimicking natural human smoking habits. Control mice were kept in a filtered air (FA) environment, but exposed to the same stress as CS-exposed animals.
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Extracted molecule |
total RNA |
Extraction protocol |
Single cells were diluted in PBS, supplemented with 0.04% bovine serum albumin up to a final concentration of 100 cells/µl. Using a microfluidic PDMS device (Nanoshift), single cells were co-encapsulated in droplets with barcoded beads (Chemgenes Corporation, Wilmington, MA) at a final concentration of 120 beads/uL. Droplets were collected for 15 min/sample. After droplet breakage, beads were harvested, washed, and prepared for on bead mRNA reverse transcription (Maxima RT, Thermo Fisher). Following an exonuclease I (New England Biolabs) treatment for the removal of unused primers, beads were treated with Klenow enzyme to improve single cell transcript quality; thereafter, beads were counted, aliquoted (2000 beads/reaction, equals ~100 cells/reaction), and pre-amplified by 12 PCR cycles (primers, chemistry, and cycle conditions identical to those previously described in Macosko et al., 2015). PCR products were pooled and purified twice using 0.6x clean-up beads (CleanNA). Prior to tagmentation, cDNA samples were loaded on a DNA High Sensitivity Chip on the 2100 Bioanalyzer (Agilent) to ensure transcript integrity, purity, and amount. For each sample, 1 ng of pre-amplified cDNA from an estimated 1500 cells was tagmented by Nextera XT (Illumina) with a custom P5 primer (Integrated DNA Technologies). Single cell libraries were sequenced in a 100 bp paired-end run on the Illumina HiSeq4000 using 0.2 nM denatured sample and 5% PhiX spike-in. For priming of read 1, 0.5 µM Read1CustSeqB was used (primer sequence: GCCTGTCCGCGGAAGCAGTGGTATCAACGCAGAGTAC).
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Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
Illumina HiSeq 4000 |
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Description |
Drop-seq muc13920
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Data processing |
For preprocessing of the single cell data of mouse lung tissue cells, the Dropseq computational pipeline was used (version 2.3.0) as previously described (Pubmed ID: 26000488). Briefly, STAR (version 2.5.3a) was used for mapping. Reads were aligned to the mm10 reference genome (GSE63269). For barcode filtering, we excluded barcodes with less than 200 genes detected. A high proportion (> 20%) of transcript counts derived from mitochondria-encoded genes may indicate low cell quality, and we removed these unqualified cells from the downstream analysis. Genome_build: mm10 Supplementary_files_format_and_content: Raw count matrix (MonocyteExtravasation_raw_counts.mtx) from Drop-seq experiments with 27780 genes and 68256 cells in market matrix format and corresponding column (MonocyteExtravasation_genes.txt) and row (MonocyteExtravasation_barcodes.txt) labels. Cell-level metadata (MonocyteExtravasxation_cells_metadata.txt).
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Submission date |
Sep 29, 2021 |
Last update date |
Dec 16, 2021 |
Contact name |
Meshal Ansari |
Organization name |
Helmholtz Zentrum Munich
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Street address |
Ingolstädter Landstrasse 1
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City |
Neuherberg |
State/province |
Bavaria |
ZIP/Postal code |
85764 |
Country |
Germany |
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Platform ID |
GPL21103 |
Series (1) |
GSE185006 |
Arginine methyltransferase regulates monocyte extravasation and function |
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Relations |
SRA |
SRX12403238 |
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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