title: GSM4063602: E18.5 CD106- RNA 2; Mus musculus; RNA-Seq
library_construction_protocol: For bulk RNAseq, lungs were removed, pooled by genotype, and minced in a petri dish on ice with a razor blade.  Minced lung tissue was incubated in 1.5% collagenase (Sigma) in TrypLE (Life Technologies) + 64U DNase I (Sigma) at 37C for 30min with occasion tituration.  The lung cell suspension was filtered successively through 100um, 40um and 20um filters, pelleted and resuspended in 20%FBS in DMEM/Hepes.  Cells were sorted on a BD FACSAria II sorter and gated for tdTomato-/GFP+ (Twist2Cre+ mesenchyme) cells.  Cells were collected, lysed in RLT+ buffer containing b-mercaptoethanol and RNA was isolated with the Nucleospin RNA Plus Kit (Macherey-Nagel).  RNAs were extracted from mesenchymal cells from Twist2Cre; For scRNAseq, Lungs were excised and placed in DMEM.  Lung lobes were diced into cubes with a microblade scapel under a dissecting microscope, placed in digest buffer (2mg/ml type2 collagenase (Worthington), 1mg/ml Pronase E (Sigma), 62.5 U/ml DNase I (Sigma), 5mM CaCl2 in PBS without calcium and magnesium) at 37C for 10min followed by gentle trituration.  Digested tissue was passed through a 23 gauge needle, filtered through a 40uM filter and cells pelleted.  Pellets were resuspended in red blood cell lysis buffer for 2 min at room temperature and washed with PBS/0.01% BSA.; For ATACseq samples, CD106+ cells were then isolated from the CD45-/CD31-/CD309- cell fraction using biotin-anti-mouse CD106 (clone 429, Miltenyi) and anti-biotin microbeads.  CD106+ cells were re-suspended in DMEM+10% FBS+25mM HEPES in Eppendorf tubes and treated with 100nM dexamethasone or saline for 4hrs at 37C.  Following treatment, cells were pelleted, counted and 50,000 cells per sample prepared for ATAC-seq. Sequencing libraries were prepared using the Nextera XT DNA Sample Preparation Kit (Illumina).
identifiers.namespace: GEO
identifiers.id: GSM4063602