px330 vector  (Addgene inc)

Journal: bioRxiv

Article Title: Epitranscriptome-wide profiling identifies RNA editing events regulated by ADAR1 that are associated with DNA repair mechanisms in human TK6 cells

doi: 10.1101/2025.07.11.664482

Figure Lengend Snippet: (A) Domain structure of the two ADAR1 isoforms, p150 and p110. (B) Schematic representation of the CRISPR/Cas9-mediated disruption of ADAR1 p150 and p150/p110. The target sequence and corresponding vectors containing either a reverse-oriented puromycin- or neomycin- resistance cassette are illustrated. (C) Western blot analysis of ADAR1 isoforms. Whole-cell extracts from WT, p150 KO clones (#1, #2, #3), and p150/p110 KO clones (#1, #2, #3) were resolved by 10% SDS-PAGE. GAPDH was used as a loading control. (D) Clustering analysis of genes exhibiting A- to-I editing. Based on inosine peak scores (fold-enrichment ratio) in WT and p150 KO cells, genes were categorized as strongly p150 dependent (WT/(p150KO+1)≧4; purple), mildly p150 dependent (11, cyan). Asterisk (*) indicates the genes categorized as p150/p110 KO-specific (WT=0, p150KO=0, p150/p110KO>1).

Article Snippet: The vectors pX330-gRNA/p150 (6 μg) and the p150 target plasmid (2 μg) were transfected into TK6 cells by a NEPA21 electroporator (Nepa Gene Co. Ltd.) following the manufacturer’s instructions.

Techniques: CRISPR, Disruption, Sequencing, Western Blot, Clone Assay, SDS Page, Control

Journal: bioRxiv

Article Title: Epitranscriptome-wide profiling identifies RNA editing events regulated by ADAR1 that are associated with DNA repair mechanisms in human TK6 cells

doi: 10.1101/2025.07.11.664482

Figure Lengend Snippet: Read coverage profiles are shown for representative DNA repair-associated genes: (A) ATM (3′UTR), (B) POLH (3′UTR), (C) POLH (intron between exon4-5), (D) ATR (intron between exon1-2), (E) FANCA (intron between exon5-6), (F), FANCA (intron between exon14-15), and (G) XPA (intron between exon5-6) in wild-type (cyan), p150 KO (pink), and p150/p110 KO cells (green). The y-axis represents read counts and the x-axis indicates genomic coordinates. The scale bar corresponds to 1 kb. “Coverage data range” in the top-right corner of each panel indicates the maximum coverage value. Colored regions within coverage tracks highlight A-to-I RNA editing sites. Strand-specific colors compositions are shown: green (Adenosine) and orange (Guanosine) for forward reads; red (Thymidine) and blue (Cytidine) for reverse reads, facilitating visual estimation of nucleotide ratios at individual position.

Article Snippet: The vectors pX330-gRNA/p150 (6 μg) and the p150 target plasmid (2 μg) were transfected into TK6 cells by a NEPA21 electroporator (Nepa Gene Co. Ltd.) following the manufacturer’s instructions.

Techniques:

Journal: bioRxiv

Article Title: Epitranscriptome-wide profiling identifies RNA editing events regulated by ADAR1 that are associated with DNA repair mechanisms in human TK6 cells

doi: 10.1101/2025.07.11.664482

Figure Lengend Snippet: (A) Gene expression levels of ATM, ATR , and FANCA were quantified by RT-qPCR in WT (black), p150 KO (gray), and p150/p110 KO (white) cells. Data represent six independent experiments (n = 6). Statistical significance was determined by Student’s t-test (p < 0.05). (B) RNA-seq read coverage profiles of the XPA gene in WT#1, WT#2, p150 KO#1, p150 KO#2, p150/p110 KO#1, and p150/p110 KO#2 cells. Arrows indicate novel splicing peaks between exon 5 and 6 of the main splicing variant. (C) Detection of XPA splicing variants. PCR was performed using cDNA from WT#1, p150 KO#1, p150 KO#2, p150/p110 KO#1, and p150/p110 KO#2, with primers targeting exons 2–6 and 5–6. Amplified products were resolved on 1.0% agarose gels. For amplification between exons 2–6, expected band sizes were 946 bp for variant 1 and 1,260 bp for a potential alternative variant. For amplification between exons 5–6, expected sizes were 544 bp for variant 1 and 964 bp for alternative variants.

Article Snippet: The vectors pX330-gRNA/p150 (6 μg) and the p150 target plasmid (2 μg) were transfected into TK6 cells by a NEPA21 electroporator (Nepa Gene Co. Ltd.) following the manufacturer’s instructions.

Techniques: Gene Expression, Quantitative RT-PCR, RNA Sequencing, Variant Assay, Amplification