Dynamic subtype- and context-specific subcellular RNA regulation in growth cones of developing neurons of the cerebral cortex

Brief Description of Contents: RNA-seq profiling of subcellular compartments of fluorescently labeled mouse callosal projection neurons (CPN) and corticothalamic projection neurons (CThPN) at two timepoints during mouse cortical development. Somata and growth cones (GC) were isolated (micro dissection; tissue dissociation; biochemical fractionation for GCs only; fluorescence-based sorting) in parallel from mouse brains at postnatal days 1 and 3. Growth cone fraction (GCF) samples were also collected post-fractionation as a baseline for comparison to GCs in order to quantify ambient RNAs. polyA+ mRNA libraries were prepared with a 1/4 volume SmartSeq v4 kit (Takara).  Data Processing Pipeline: 1. Adapters were trimmed and low-quality reads were filtered using the TrimGalore! wrapper for Cutadapt 2. Sequences aligning to rRNA, rodent repeat elements, 7SL or SRP (the RNA component of the signal recognition particle), or the mitochondrial genome were removed using bbsplit.sh from the BBTools suite 3. Reads were aligned to the GRCm38/mm10 genome and to the corresponding Ensembl transcriptome version 101 using STAR (version 2.7.9a) with default parameters. 4. Transcript abundances were estimated using Salmon (version 1.7.0) alignment mode with [ –posBias –numBootstraps 100] flags set. 5. Differential expression at the gene level was calculated using DESeq2 (version 1.26.0); gene abundances were estimated using aggregated transcript counts. 6. Due to 3'bias in the data, differential expression at the transcript level was calculated by first identifying and counting only reads overlapping with the last 250bp of each transcript, and then using DESeq2 (version 1.26.0).