Experimental overview and characterization of the comprehensive transcriptome and the cytosol. (A) Schematic of the experiment and subsequent bioinformatic analysis workflow of the resulting cytosolic extract and fractionated, ribosome-associated short and long reads from ESCs and NPCs. (B) The transcriptome classified by SQANTI3-defined structural categories of spliced transcripts, including: full splice match (FSM), incomplete splice match (ISM), novel in catalog (NIC), novel not in catalog (NNC), and intergenic or fusion transcripts (other). FSM and ISM transcripts match annotated splice sites and junctions (in GRCh38.p13 release 41), whereas NIC transcripts comprise novel combinations of annotated splice sites and junctions, and NNC transcripts contain at least one unannotated splice site. (C) The transcriptome classified by productivity based on the detection of complete or incomplete open reading frames (productive or noncoding respectively), premature stop codons (NMD), and retained introns (RIs). (D) Stratification of the transcriptome by the number of isoforms and unique coding sequences per gene. (E,F) Gene-level (E) and transcript-level (F) differential expression between NPC and ESC cytosolic fractions. (G) Top 12 enriched Metascape pathways in differentially expressed genes between NPC and ESC cytosolic fractions.