GSM9153880: EOL1_rep2; Homo sapiens; RNA-Seq
The lungs, trachea, and bronchus were cut into small pieces with surgical scissors and incubated with a protease mixture composed of 4 U/ml elastase, 5 U/ml dispase, 450 U/ml collagenase type III, and 200 µg/ml DNase in RPMI-1640 for 30 min at 37 °C with agitation. The digested lungs were then resuspended with a trypsin mixture consisting of TrypLE Express and 80 µg/ml DNase, incubated at 37 °C for 5 min with frequent agitation, and then washed with 10% fetal bovine serum RPMI. Cell suspensions were incubated with anti-CD16/CD32 to block Fc receptors for 15 min at 4 °C. Whole mouse lung cells were freshly prepared, and for PNEC accumulation, EpCAM+ CD45- tdTomato+ cells and EpCAM+ CD45- tdTomato- cells from PNECs reporter (Ascl1-CreERT2::Rosa26-tdTomato) mice were sorted by BD FACS Aria III. For EoL-1 cells, all cells were recovered for RNA with Trizol and RNA was recovered by the chloroform-phenol method. Library was prepared according to the manufacter's instructions (Chromium Next GEM Single Cell 3' Reagent Kits v3.1, Dual Index). Briefly, the cells were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion. The poly-A RNA from the cell lysate contained in every single GEM was retrotranscripted to cDNA, which was contains an Illumina R1 primer sequence, Unique Molecular Identifier and the 10x Barcode. The pooled barcoded cDNA was then cleaned up with Silane DynaBeads, amplified by PCR and the apropriated sized fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction Illumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added. RNA-Seq was performed as previously described49. Total cellular RNA was extracted using TRIzol reagent (Invitrogen). A SMARTer Stranded Total RNA-Seq Kit v2 pico (Takara Bio Inc., Shiga, Japan) was used according to the manufacturer's protocol for cDNA library construction. Sequencing of library fragments was performed using an Illumina NextSeq500 System.