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| Status |
Public on Sep 10, 2021 |
| Title |
TG_1-transgene-am-RNA |
| Sample type |
SRA |
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| Source name |
airway macrophages
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| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
genotype: Scnn1b-transgene
tissue: lung
cell type: airway macrophages
batch: 1
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| Growth protocol |
One ml dispase (BD Biosciences, Heidelberg, Germany) was intratracheally instilled into the lungs and plugged inside by instillation of 300 ml low melting agarose (1%). When agarose is solid, whole lung was extracted including the tracheobronchial tree, put into 2 ml dispase and left for 35 min at RT. Reaction was stopped with complete DMEM, lungs were mashed with a plunger, tracheobronchial tree was removed and cell suspension put through a 100 mm cell strainer (BD Biosciences, Heidelberg, Germany). After red blood cell lysis (RBC lysis buffer, eBiosciences, Dreieich, Germany) cell suspension was enriched by magnetic bead separation using CD45+ magnetic beads, according to manufacturer’s instructions (Miltenyi Biotech, Bergisch Gladbach, Germany). After positive selection cells were counted and antibody-concentration used according to obtained cell numbers, as prior determined by titration. Cells were incubated for 5 min with purified rat IgG2b anti-mouse CD16/CD32 receptor antibody (BD Biosciences, Heidelberg, Germany) in FACS buffer containing 1 % BSA (SERVA Electrophoresis GmbH, Heidelberg, Germany) and 5 mM EDTA (Sigma-Aldrich, Darmstadt, Germany), following staining with fluorochrome-conjugated antibodies against murine CD45.2, Siglec-F and CD11c (see Supplementary table 5). Samples were stained with 7-AAD (0.25 mg/ml per 1x106 cells; Biolegend, London, UK) 5min prior to sort, for dead cell exclusion. Sorting was performed at the EMBL Flow Core Facility, Heidelberg, Germany. AMs were sorted using a standard BD Fusion equipped with 100-mW 405-nm, 100-mW 488-nm, 80-mW 561-nm, 80-mW 640-nm lasers and an ND2.0 filter in front of the FSC photodiode, a nozzle size of 100 μm, and corresponding BD FACSFlow sheath pressure of 20 psi, matched with a transducer frequency of 32 kHz. Input pressure was adjusted to ensure that every fifth to sixth drop was populated by an event. Purity check of sorted cells was performed on selected samples from each run confirming purities ranging 95-99 %.
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| Extracted molecule |
total RNA |
| Extraction protocol |
DNA and RNA were isolated using TRIzol reagent (Sigma Aldrich, Darmstadt, Germany) and further AllPrep DNA/RNA Mini Kit (Qiagen, Hilden, Germany) purification, following the manufacturer’s instructions. For RNA isolation (RNase-free DNAse set, Qiagen) DNAse I digestion was performed on the column. Proteinase K digestion was applied for RNA and DNA isolation (Puregene Proteinase K, Qiagen). Prior to library preparation, DNA and RNA quality was checked by Agilent Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) and quantified by Qubit fluorimetry (Thermo Scientific, Wilmington, USA). All RNA samples reached an RNA integrity number (RIN) > 8.5.
For the paired-end baseline as well as LPS- and medium-treated replicates of wt- and tg-AM, sequencing libraries were prepared by the DKFZ Genomics and Proteomics Core Facility from total RNA using the SMART-Seq v4 Ultra Low Input RNA Kit according to the manufacturer’s instructions. For sequencing, samples were sequenced on a HiSeq 2000 v4, paired-end, 125 bp platform (Illumina). Single-end baseline replicates were prepared by the EMBL Genomics Core Facility from total RNA using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina. Sequencing was performed on a Next Seq 500, single-end, 75 bp platform.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
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| Data processing |
RNA-seq data was processed with the nf-core RNA-seq pipeline (Ewels et al., 2020) (v. 1.2). Default parameters were used unless mentioned otherwise. Sequences were aligned to the mouse reference genome mm10 by application of the software HISAT2, with --unstranded option. For single-end data the -singleEnd option was applied. Transcripts were assembled using StringTie and gene code gene annotation release M20 (Frankish et al., 2019). Gene counts were generated with Stringties prepDE.py script (setting: -eb).
For the identification of differentially expressed genes, the R package DESeq2 was used (Love, Huber, & Anders, 2014). For group-wise comparison of baseline replicates, the littermate was included in the design formula to adjust for batch effects. For the LPS stimulation experiment the date of library preparation was included. The adjusted p-value as well as log2 fold change required to fulfil statistical significance is mentioned in the figure legends.
Genome_build: mm10
Supplementary_files_format_and_content: Count file as tab-delimited text file. Excel sheet containing differentially expressed genes; Scnn1b-transgene vs wildtype at baseline
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| Submission date |
Jul 21, 2020 |
| Last update date |
Sep 10, 2021 |
| Contact name |
Joschka Hey |
| E-mail(s) |
joschkahey@gmail.com
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| Organization name |
DKFZ
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| Department |
Cancer epigenomics
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| Lab |
Plass lab
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| Street address |
Im Neuenheimer Feld 280
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| City |
Heidelberg |
| State/province |
Deutschland |
| ZIP/Postal code |
69120 |
| Country |
USA |
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| Platform ID |
GPL13112 |
| Series (2) |
| GSE154805 |
Epigenetic reprogramming of airway macrophages drives polarization and inflammation in muco-obstructive lung disease (RNA_BL) |
| GSE154808 |
Epigenetic reprogramming of airway macrophages drives polarization and inflammation in muco-obstructive lung disease |
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| Relations |
| BioSample |
SAMN15590000 |
| SRA |
SRX8783693 |
| 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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