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| Status |
Public on Sep 10, 2021 |
| Title |
TG_15-transgene-am-ATAC |
| 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: 3
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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 |
genomic DNA |
| 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.
ATAC libraries have been prepared according to the Omni-ATAC protocol (Corces et al., 2017a) with minor modifications. In short, 50000 viable cells were pelleted and wahsed in PBS. Subsequently, nuclei were isolated using cold lysis buffer (0.01% Digitonin [Sigma-Aldrich, Munich, Germany], 1% NP40 [Genaxxon Bioscience, Ulm, Germany], 0.1% Tween-20 [Sigma-Aldrich, Munich, Germany]). Resuspension of the nuclei was done in ATAC washing buffer (10 mM Tris-HCl pH 7.4 [Sigma-Aldrich, Munich, Germany], 10 mM NaCl [Sigma-Aldrich, Munich, Germany], 3 mM MgCl2 [Sigma-Aldrich, Munich, Germany]).The tagmentation reaction was performed in 2x transposition buffer (20 mM Tris-HCl pH 7.6 [Sigma-Aldrich, Munich, Germany], 3 mM MgCl2 [Sigma-Aldrich, Munich, Germany], 20% Dimethyl Formamide [Sigma-Aldrich, Munich, Germany]) by the addition of 2.5µl of Tagment DNA Enzyme 1 (Illumina, San Diego, CA) and rotating the mixture at 1000 rpm for 30 minutes at 37°C. The reaction was stopped by the addition of 20µl of 5M Guanidinium thiocyanate (Sigma-Aldrich, Munich, Germany). Transposed chromatin was then purified using 30µl of AMPure XP beads beads (Beckman Coulter, Brea, CA)), and 110 µl of PEG buffer (2.5 M NaCl [Sigma-Aldrich, Munich, Germany], 20% PEG 8000 [Sigma-Aldrich, Munich, Germany]). Library amplification was separated in a two-step procedure. First, 25 µl of NEBNext High Fidelity 2x Master Mix (NEB, Ipswich, MA), 0.8 µl of 10 µM Custom Nextera PCR Primer 1, and 0.8 µl of 10 µM Custom Nextera PCR Barcode was added to 25µl of the transposed DNA, using the following program: 5 minutes at 72°C, 30 seconds at 98°C, 5 cycles of 10 seconds at 98°C, 30 seconds at 63°C, and 1 minute at 72°C, and, finally, 1 minute at 72°C. 5µl of the pre-amplified PCR mixture were used to determine how many additional cycles were needed to reach sufficient amplification of each library. For this, Sybr Green I nucleic acid gel stain (Thermo Fisher Scientific, Waltham, MA) was added and quantitative PCR performed with light cycler instrument (Roche, Basel, Switzerland) by applying the following program: 30 seconds at 98°C, 20 cycles of 10 seconds at 98°C, 30 seconds at 63°C, 1 minutes at 72°C, and, finally, 1 minute at 72°C. In a second step, the previous identified PCR cycles were applied to the remaining pre-amplified mixture. The libraries were then purified with a left sided size selection applying 1.4x of AMPure XP beads (Beckman Coulter, Brea, CA) and finally resuspended in 1x elution buffer (Qiagen, Hilden, Germany). Fragment size distribution was checked by Agilent Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) and concentrations were quantified by Qubit fluorimetry (Thermo Scientific, Wilmington, USA). Sequencing was performed in multiplexes at the DKFZ Genomics and Proteomics Core Facility using the HiSeq 2000 v4 paired-end 125 bp platform (Illumina).
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| Library strategy |
ATAC-seq |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2000 |
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| Data processing |
Processing of the ATAC-seq reads were performed using the ENCODE ATAC-seq pipeline (https://github.com/ENCODE-DCC/atac-seq-pipeline; 10.5281/zenodo.156534.svg) using default parameters. As a reference genome mm10 was used. Each baseline replicate achieved a minimum of 50 million non-duplicated, non-mitochondrial reads. The irreproducible discovery rate was less than two for each group of replicates and the fraction of reads in called peak regions above 0.5.
Differential accessibility was performed using the R package DiffBind (Stark, 2011). In short, a common peak set was identified by the presence of a peak in at least two samples. As a method for differential analysis edgeR was further applied (Robinson, Mccarthy, & Smyth, 2010). For the LPS stimulation experiment the date of RNA isolation was included as a blocking factor to adjust for batch effects. To define the LPS treatment effect in Fig. 4, count values were extracted for every consensus peak and a multi-factor design including the batch, genotype, treatment and interaction term (~batch+genotype+treatment+genotype:treatment) was constructed using DESeq2. Only the treatment covariate was extracted using the following contrast: c("treatment","medium", "LPS"). LPS responsive regions with an adjusted p-value <0.05 and a log2 fold-change > 2 were considered as significant. Annotation of all differentially accessible regions was performed with the R package ChIPseeker (Yu, Wang, & He, n.d.) and TxDb.Mmusculus.UCSC.mm10.knownGene (BC, 2019).
Genome_build: mm10
Supplementary_files_format_and_content: Excel file with differentially accessible regions; 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) |
[email protected]
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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) |
| GSE154804 |
Epigenetic reprogramming of airway macrophages drives polarization and inflammation in muco-obstructive lung disease (ATAC_BL) |
| GSE154808 |
Epigenetic reprogramming of airway macrophages drives polarization and inflammation in muco-obstructive lung disease |
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| Relations |
| BioSample |
SAMN15590002 |
| SRA |
SRX8783750 |
| 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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