GSM4876743: 2252019_3; Homo sapiens; RNA-Seq
Freshly brush-biopsied human bronchial samples and micro-dissected bronchioles were incubated with Accutase solution for 30 min at 37°C. The Accutase-treated cells were centrifuged (600 g, 2 min, 4°C) and then incubated with 10 mL HBSS (Ca+, Mg+) buffer containing DNase I (0.1 mg/ml) and collagenase IV (1 mg/ml) for 10 min at 37°C. After incubation, collagenase IV was inactivated by adding 500 μL fetal bovine serum. Dissociated cells were filtered through a 40-μm cell strainer, centrifuged (600 g, 2 min, 4°C) and resuspended in PBS, adjusted to 105 cells/ml. Cell viability was examined by trypan blue dye exclusion. single cell-based RNA libraries were prepared for sequencing using standard Drop-Seq library preparation protocols (Macosko EZ, et al., Cell 2015; 161: 1202-1214). The libraries were sequenced using HiSeq X with 150-bp paired-end sequencing.