Illumina NovaSeq 6000 paired end sequencing; RNAseq of proximal and distal murine lung epithelial cells isolated with 3DLD before and after differentiation
RNAseq of proximal and distal murine lung epithelial cells isolated with 3DLD before and after differentiation
3DLD isolation method was used to isolate both proximal and distal progenitors from individual mice. Proximal epithelial cells were expanded to passage 1 and collected by pelleting the cells. Distal progenitors from 3DLD isolation or Classic isolation were directly pelleted after isolation protocol. Qiagen RNeasy micro kit (74004, QIAGEN, Sweden) was used to isolate total RNA following the manufacturer's protocol. RNA concentrations were measured using NanoDrop 1000 and Qubit™ RNA HS Assay Kit (Q32852, Fisher Scientific). RNA quality was evaluated by the agilent 2100 BioAnalyzer.  Library preparation was done using the Illumina Stranded mRNA Prep Ligation (20040534, Illumina) with 14 cycles under final PCR amplification and samples were indexed with IDT for Illumina RNA UD Indexes Set B, Ligation (20040554, Illumina) for multiplexing. Clean up steps were automated using the King Fisher FLEX (18-5400620, Thermo Scientific). Library concentration was checked using the QuantIT 1X dsDNA HS Assay Kit (Q33232) and library size and quality were checked using the 5200 Fragment Analyzer™ 12-capillary (M3510AA, Agilent) using the DNF-930 dsDNA Reagent Kit, 75 bp – 20,000 bp(500 Samples) (Part # DNF-930-K0500, Agilent).