title: GSM8145466: C1 (GFP-low, TdT-neg); Mus musculus; RNA-Seq
library_construction_protocol: Lungs were minced with a razor blade with 50 µl of dispase (5,000 Caseinolytic Units, Discovery Labware, Inc. 354235) and then transferred to PBS containing 833 units /ml dispase, 0.01 mg/ml DNase I (Roche; 10104159001) and 2 mg/ml collagenase/dispase (Roche, 10269638) at 37°C for 40 min with gentle agitation. Digestion enzymes were then inactivated with FACS buffer (10% FBS, 1 mM EDTA in PBS), samples were sequentially filtered through a 70 µM cell strainer (Thermo Fisher Scientific; 22363548), and then a 40 µM cell strainer (Thermo Fisher Scientific; 22363547), and then centrifuged at 800 x g for 4 min. Red blood cells were lysed with ACK lysis buffer (Gibco; A10492-01) and cells were resuspended in FACS buffer. SYTOX blue (1 µl, Thermo Fisher Scientific, S34857) was added to each tube for live/dead sorting. Cells were sorted using a SY3200 “Synergy” cell sorter (Sony Biotechnology) or a Beckman Coulter MoFlo cell sorter (Beckman Coulter Life Sciences) with a 100 µm nozzle directly into FACS buffer or culture media (1.5 ml) on ice. Four cell populations were collected: TDTomato+, GFP-; TDTomato+, GFPHigh; TDTomato-, GFPHigh; and TDTomato-, GFPLow. Single channel controls (Fgf18CreERT2; ROSATDTomato; PdgfrαEGFP mice) were used to set compensation at the time of sorting and fluorescence minus one control (ROSATDTomato , lacking Fgf18CreERT2) was used as a negative control.  Plotted percentage of the four populations of cells was calculated relative to the total number of sorted cells. Libraries were constructed according to manufacturer's protocol (10X Genomics, 3'v3.1). Cells were loaded in master mix with partitioning oil and beads to a chip and GEMs formed using a 10X Chromium device. Reverse transcription was performed to create barcoded cDNA from poly-A RNAs in each GEM. Pooled cDNA was cleaned with Silane Dynabeads, amplified, and size-selected using SPRIselect. Illumina R2 primer sequence was added along with paired-end constructs with P5/P7 sequences and a sample index for sequencing.