GSM5208110: fetal lungs, Combo-blockade-3; Macaca mulatta; RNA-Seq
The fetal lung was removed, and left lobe was processed into single cell suspensions as described previously 56. The large airways were removed and approximately ~500mg of airway free tissue was collected in a gentleMACS C-tube (Miltenyi Biotec, Auburn, CA) and 5mL digest buffer was added and placed on a gentleMACS octo dissociator for programmed runs. Following dissociation, the cell suspension was passed through a 100µm filter and washed with PBS. After washing, cells underwent red cell lysis (eBioscience, San Diego, CA) along with another pass through a 40um filter after neutralization. ~50,000 cells per lung were submitted for single cell sequencing at the Cincinnati Children's Hospital Medical Center DNA Sequencing and Genotyping Core. Approximately ~16,000 cells were loaded into one channel of the Chromium system using the 3 prime v3 single cell reagent kit (10X Genomics, Pleasanton, CA). Following capture and lysis, cDNA was synthesized and amplified as per the manufacturer's protocol (10X Genomics). The amplified cDNA was used to construct Illumina sequencing libraries that were each sequenced using an Illumina HiSeq 4000 machine.