nebnext ultra rna library prep kit  (New England Biolabs)

Journal: bioRxiv

Article Title: Neuronal late endosomes serve as selective RNA hubs disrupted by ALS-linked FUS mutation

doi: 10.64898/2026.01.19.700337

Figure Lengend Snippet: Proximity labeling reveals a distinct transcriptome associated with RAB7A-positive endosomes in motor neurons. (A) Schematic representation of the APEX2-based proximity labeling strategy used to biotinylate RNAs in the vicinity of RAB7A-positive endosomes in iPSCs-derived MNs. (B) Western blot analysis of the APEX2-pulldown on iPSCs-derived MNs expressing flagged APEX2-RAB7A protein. Early endosomal markers (EEA1, RAB5), Late endosomal marker (RAB7A), Late endosomal/lysosomal marker (LAMP1), APEX2-RAB7A construct (FLAG), Nuclear marker (H3), and mitochondrial marker (TOM20) were used to assess compartment purification. INP = 2,5% input, PD = pull down, H2O2= hydrogen peroxide treatment. (C) Volcano plot showing the log2FC pull-down/input and the −log10Pvalue for each RNA detected. Enriched and Depleted RNAs (Padj < 0.05, |log2FC| > 0.59) or Unspecific RNAs are indicated by red, blue and gray dots, respectively. (D) Dotplot depicting the cellular component GO over-represented categories of the Enriched RNAs. Only significant categories (FDR < 0.05) were depicted. X-axis represents category enrichment score, while Y-axis reports the GO category description. Dot size represents the amount of RNAs in the analyzed group that overlap the category. (E) Representative RNA FISH and immunofluorescence images showing localization of an endosome- enriched (DNAJC5) or endosome- depleted (ARGLU1) mRNA (magenta) relative to RAB7A-positive endosomes (cyan) in soma and neurites. Insets show 3D renderings of boxed regions. Scale bar = 5 μm. Colocalization is indicated by yellow arrows. (F) Quantification of the percentage of endosome- enriched (red), unspecific (gray), or depleted (blue) RNAs associated with RAB7A-positive particles. Data are shown as boxplots; p-values are indicated with asterisks (**p<0.01, ***p<0.001, ****p<0.0001). Statistical significance was assessed with One-way Brown-Forsythe and Welch ANOVA tests. N = 3 biological replicates (total cells: NORAD n = 315, DNAJC5 n = 270, TMOD2 n = 278, CACNG4 n = 272, LRRC4B n = 295, AGPAT4 n = 269, ARGLU1 n = 274).

Article Snippet: Total RNA was isolated using the Direct-zolTM Miniprep RNA Purification Kit (Zymo Research), including an on-column DNase digestion step, according to the manufacturer’s instructions.

Techniques: Labeling, Derivative Assay, Western Blot, Expressing, Marker, Construct, Purification, Immunofluorescence