GSM4514137
GSM4514137: 10X_2-4mHet_3; Mus musculus; RNA-Seq
For harvesting, mice were euthanized by CO2 asphyxiation followed by exsanguination.  Lungs were perfused with chilled PBS via the right heart.  For lung cells isolation without enrichment (15 out of 18 samples), dispase (50 U-mL) was instilled into lungs, which were dissected and minced on ice into approximately 1 mm3 cubes.  Lungs were digested using collagenase-dispase for 30 mins at 37°C, then passed through a 40 um cell strainer.  Cells were centrifuged at 300 RCF for 5 mins, then incubated with RBC lysis buffer.  Cells were stained with trypan blue for viability assessment which showed greater than 85% viability with this method. The single cell suspension was sorted by FACS for DAPI-negative viable cells using MoFlo Astrios EQ. For enriched CD45- lung cells (4mHet-3, 4mWt-2, 8mHet-3), lungs were digested using a mouse lung isolation kit with gentleMACS dissociator (Miltenyi), then were passed through a 40 um cell strainer. After centrifugation at 300 RCF for 5 mins, cells were incubated with RBC lysis buffer. Cells were then stained with DAPI, DRAQ5 (abcam) and CD45-PE Ab (BioLegend), and selected for DAPI negative, DRAQ5 positive and CD45 negative cells using On-chip Sort cell sorter (On-chip Biotechnologies)(Watanabe et al., 2014). single cell RNA libraries were prepared for sequencing using 10X Genomics Chromium V2 protocols and inDrops
GEO
GSM4514137
GEO Accession
GSM4514137