kapa rna hyperprep kit with riboerase hmr  (Roche)

Journal: iScience

Article Title: Heterozygous ADAR mutant mice exhibit RNA sensing-dependent neuroinflammation and phenotypes associated with Aicardi-Goutières syndrome

doi: 10.1016/j.isci.2026.114758

Figure Lengend Snippet: RNA editing and protein level changes caused by the G1007R mutation (A) RNA editing levels in well-defined RNA editing sites. Data were assessed by RT-PCR Sanger sequencing on brain RNAs from WT and Adar WT/G1007R mice. Editing levels at the A site in Htr2c mRNA and editing in mRNAs of Blcap and Ube2o are modestly decreased, while editing at other sites are not different from the WT controls, and the D site in the Htr2c mRNA, known to be the ADAR2 editing site, is increased. Data are presented as mean ± SD, n = 4 for both WT and Adar WT/G1007R mice. The Prism t test was used to test the differences between the two groups. ∗ p < 0.05, ∗∗∗ p < 0.001. (B) RNA editing site numbers in WT and Adar WT/G1007R mice. Data were assessed through deep RNA-seq data analysis. The total numbers of RNA editing sites in each brain samples were averaged in the WT and Adar WT/G1007R groups. The numbers of editing sites are not significantly different between the two groups. n = 3 for both WT and Adar WT/G1007R groups. The Prism t test was used to test the differences between groups. The average editing levels are not different. (C) Overall averaged RNA editing levels are shown. Three mice in each group were assessed from the RNA-seq data. A value of 1.0 indicates 100% editing. (D) RNA editing levels at editing sites in repetitive sequences. The averaged value of the editing sites in the WT and the Adar WT/G1007R mice are shown. There is no significant difference between the WT and Adar WT/G1007R mice. n = 3 for both WT and Adar WT/G1007R groups. The Prism t test was used to test the differences between the two groups. (E) RNA editing efficiency at individual editing sites. Sites include the well-defined editing sites in Gria2 , Gria3 , Griak , and Htrc2 mRNAs, and data were calculated from the RNA-seq data. Editing at the known editing sites for ADAR1, including the A and B sites in Htrc2 mRNA, in the Blcap , Neil1 , Ube2o mRNAs was significantly decreased. n = 3 for both WT and Adar WT/G1007R groups. The Prism t test was used to test the differences between groups. ∗ p < 0.05, ∗∗ p < 0.01. (F) ADAR1 protein levels in the brain from individual mice. Data were assessed by western blot with a specific antibody recognizing both the p150 and p110 isoforms of ADAR1. The p110 isoform is predominantly expressed in the brain. The ADAR1 protein level is dramatically decreased in the Adar WT/G1007R mice. (G) Averaged ADAR1 protein levels for WT and Adar WT/G1007R mice. Protein level of p110 isoform was quantified by the density of the protein signals on western blots from independent experiments. The ADAR1 protein level in the Adar WT/G1007R mice is significantly lower than the WT controls. Data are presented as mean ± SD, n = 6 for both WT and Adar WT/G1007R groups. The Prism t test was used to test the differences between groups. ∗∗∗ p < 0.001.

Article Snippet: Nine independent RNA libraries were generated using the KAPA RNA HyperPrep Kit with the removal of ribosome RNAs by RiboErase (Kapa Biosystems) according to the manufacturer’s protocol, and the libraries were sequenced using the NovaSeq6000 platform (Illumina) to an average of 80M 10o bp paired end reads.

Techniques: Mutagenesis, Reverse Transcription Polymerase Chain Reaction, Sequencing, RNA Sequencing, Western Blot

Journal: iScience

Article Title: Heterozygous ADAR mutant mice exhibit RNA sensing-dependent neuroinflammation and phenotypes associated with Aicardi-Goutières syndrome

doi: 10.1016/j.isci.2026.114758

Figure Lengend Snippet: The G1007R mutation causes alternative splicing in Adar G1007R/G1007R mice (A) ADAR1 protein levels in embryos. Total protein from E11.5-day embryos of Adar WT/G1007R mating was analyzed by western blot with anti-ADAR1 antibody. The p110 isoform of ADAR1 is expressed in the WT embryos, and the p150 isoform is undetected. The p110 isoform in Adar G1007R/G1007R embryos is dramatically reduced compared to WT, while the p150 isoform is detectable. Quantification of p150 and p110 isoforms is shown on the right, showing dramatically reduced p110 in Adar G1007R/G1007R embryos. Data are presented as mean ± SD, n = 3. The Prism t test was used to test the differences between the two groups. ∗∗∗ p < 0.001. (B) ADAR1 protein levels in brain. Total protein from brains was isolated from two-week-old mice. The p110 isoform of ADAR1 is detected in both the mutant and WT mice, but the quantity in the mutant mice is much less, while the p150 isoform was not detected. Quantification of p110 isoform is shown on the right, showing dramatically reduced p110 in Adar G1007R/G1007R ; Ifih1 −/− brains. Data are presented as mean ± SD, n = 3. The Prism t test was used to test the differences between the two groups. ∗∗∗ p < 0.001. (C) cDNA alignment shows altered sequences. The cDNA clones of the E11.5-day Adar G1007R/G1007R embryonic total RNAs were subjected to Sanger sequencing. The cDNA sequences covering the region of exon 10 to exon 13 are compared between the Adar G1007R/G1007R and WT embryos . The sequences of the corresponding exons are indicated by alternative colors. The G>A mutation site is highlighted in red and boxed. The dashes indicate the missing sequences, including the missing exon 11, exon 12, or the 27 nucleotides in 3′end of exon 11. The seven extra nucleotides highlighted in blue are from intron 11. Five different mRNA sequences containing different exons or intron are shown here. (D) The genetic G>A mutation (G1007R) at 3′ end of exon 11 in the Adar gene alters the TG splice donor site sequence to TA. A chromatogram from Sanger sequencing of a heterozygous genome flanking the mutation site is shown. The donor site is boxed, and the mutation is indicated by an arrow.

Article Snippet: Nine independent RNA libraries were generated using the KAPA RNA HyperPrep Kit with the removal of ribosome RNAs by RiboErase (Kapa Biosystems) according to the manufacturer’s protocol, and the libraries were sequenced using the NovaSeq6000 platform (Illumina) to an average of 80M 10o bp paired end reads.

Techniques: Mutagenesis, Alternative Splicing, Western Blot, Isolation, Clone Assay, Sequencing

Journal: iScience

Article Title: Heterozygous ADAR mutant mice exhibit RNA sensing-dependent neuroinflammation and phenotypes associated with Aicardi-Goutières syndrome

doi: 10.1016/j.isci.2026.114758

Figure Lengend Snippet: Alternative splicing caused by the G1007R mutation (A) Comparison of normal splicing (splicing 1, SP1, between exon 11 and 12) to identified variants. The other four splicing variants (SP2-5) do not occur at the donor site of exon 11. The genome structure from exon 10 to 13 at the Adar locus, along with the splicing site sequences, is shown here. The A>G mutation at the donor site of exon 11 is highlighted in red. The −27 splicing site in Exon 11 and the +7 splicing site in intron 11 are indicated. Splicing pattern 1 (SP1) comprised intact exon 11 and exon 12. Splicing patterns 2–5 skip exons or use alternative splicing sites, generating RNA transcripts of different lengths. (B) Sequences surrounding the splicing sites of the five alternative splicing patterns. Exonic sequences are indicated in red, while intronic sequences are shown in blue. The seven nucleotides of intron 11 are shaded, and the splicing donor sites are boxed. The corresponding Sanger sequencing results of the cDNA clones of the alternatively spliced mRNAs are presented in . (C) Comparison of WT and Adar G1007R/G1007R ; Ifih1 −/− RNA transcripts. The total RNA-seq reads transcribed from exon 10–13 at Adar locus are compared between the WT and Adar G1007R/G1007R ; Ifih1 −/− mice at two weeks of age. There is significantly less RNAs transcribed from the G1007R mutant Adar alleles. Data are presented as mean ± SD, n = 3, and the Prism t test was used to test the differences between the two groups. ∗ p < 0.05. (D) The numbers of the spliced and non-spliced RNAs were compared between the WT and Adar G1007R/G1007R ; Ifih1 −/− mice. The number of the spliced RNAs is dramatically less in the Adar G1007R/G1007R ; Ifih1 −/− mice than the WT mice, while there are more non-spliced RNAs (contain intron sequences) in the G1007R mutant mice. Data are presented as mean ± SD, n = 3, and the Prism t test was used to test the differences between the two groups. ∗∗∗ p < 0.001. (E) Alignment of the spliced RNAs in RNA-seq data mapping to exons 10–13 of Adar . RNA reads from one of the Adar G1007R/G1007R ; Ifih1 −/− mouse is shown. The colored boxes represent the RNA sequences and lines represent the intron sequences that do not present in the RNA reads. Different colors show the various splicing patterns. (F) Relative quantities of each spliced type. Data were calculated according to the numbers of each spliced RNA read in three Adar G1007R/G1007R ; Ifih1 −/− mice. The average of the percentile of each splicing type is shown. The splicing between exon 11 and 12 is the most frequent, followed by the splicing skipping exon 11. The splicing skipping of exons 11 and 12 is the least frequent among these five alternative splicing patterns. The −27 and +7 splicing comprise less than 20% of the total spliced RNAs for each splicing type. (G) Comparison of ADAR1 RNAs spliced at exon 11 and 12 (SP1) between the WT and Adar G1007R/G1007R ; Ifih1 −/− mice. SP1 is significantly decreased in Adar G1007R/G1007R ; Ifih1 −/− mice than the WT controls. Data are presented as mean ± SD, n = 3, the Prism t test was used to test the differences between the two groups. ∗∗∗ p < 0.001.

Article Snippet: Nine independent RNA libraries were generated using the KAPA RNA HyperPrep Kit with the removal of ribosome RNAs by RiboErase (Kapa Biosystems) according to the manufacturer’s protocol, and the libraries were sequenced using the NovaSeq6000 platform (Illumina) to an average of 80M 10o bp paired end reads.

Techniques: Alternative Splicing, Mutagenesis, Comparison, Sequencing, Clone Assay, RNA Sequencing