GSM6467312: WT Male_RoomAir; Mus musculus; RNA-Seq
The right ventricle was perforated and lungs were perfusedwith 5 ml of DPBS until completely white. Lungs were removed, and shortly rinsed with DPBS (Lonza). Dissected lungs were then digested using the Miltenyi Biotec Lung Dissociation Kit (130-095-927) at 37 °C and the gentleMACS™  Dissociator (Miltenyi Biotech, Germany) using the program  gentleMACS Program m_lung_02. The suspension was then treated with red cell lysis buffer and passed throuh a 70um stariner. The cells were stained with DAPI and subjected to flow cytometric analysis to collect DAPI negative viable cells for downstream single-cell analysis. Single cell Gene Expression Library was prepared according to Chromium Single Cell Gene Expression 3v3.1 kit (10x Genomics). In Brief, single cells, reverse transcription (RT) reagents, Gel Beads containing barcoded oligonucleotides, and oil were loaded on a Chromium controller (10x Genomics) to generate single cell GEMS (Gel Beads-In-Emulsions) where full length cDNA was synthesized and barcoded for each single cell. Subsequently the GEMS are broken and cDNA from each single cell are pooled. Following cleanup using Dynabeads MyOne Silane Beads, cDNA is amplified by PCR. The amplified product is fragmented to optimal size before end-repair, A-tailing, and adaptor ligation. Final library was generated by amplification.