Comparative translating ribosome affinity purification-RNAseq (TRAPseq) by lung cell-specific drivers
Translating ribosome affinity purification coupled to next-generation RNA sequencing permits profiling of specific cellular compartments. Here, we used well-characterized Cre and CreER drivers to lineage trace different cell populations in the developing lung with a ribosomal L10-GFP fusion protein. Two different anti-GFP antibodies were used to pull down ribosomes. mRNA was isolated, reversed transcribed, and processed using next-generation sequencing. Profiling showed distinct clustering between alveolar cell drivers and epithelial cell drivers, which were clearly distinguishable from raw mRNA input. Overall design: 49 samples are included here -- 3 of raw input (supernatant from purification) and 46 TRAP-seq specimens. These include 16 cell-specific Cre and CreER drivers specific to alveolar, epithelial, smooth muscle, or other cell types with two different anti-GFP  antibodies. Neonatal lungs (~P5) were dissected and stripped from surrounding tissue. Translating ribosomes were isolated per previous protocol (see for instance 10.1242/dev.160788), and pulled down using one of two anti-GFP antibodies (19C8 or Rb). cDNA libraries constructed using 300 bp inserts and submitted for next-gen reads on Illumina NextSeq 500 pipeline. Raw reads processed for QC using fastqc and poor quality reads filtered using trimmomatic. Aligned to mm10 genome using STAR aligner and counts quantitated, normalized, and all pairwise comparisons done using DESeq2.