Journal: Genome Research

Article Title: Methyl-SNP-seq reveals dual readouts of methylome and variome at molecule resolution while enabling target enrichment

doi: 10.1101/gr.277080.122

Figure Lengend Snippet: Overview of Methyl-SNP-seq. ( A ) Experimental workflow of Methyl-SNP-seq: (1) The genomic DNA is fragmented to 300–400 bp fragments. (2) Hairpin adapters are ligated at both ends of the fragmented DNA, forming a dumbbell-shaped DNA. Next, nicks at both opposite ends of the adapters are introduced and using nick translation, a copy of the original strand is synthesized, replacing CTP as a source of nucleotide with m5CTP instead. This nick translation step breaks the dumbbell-shaped DNA somewhere within the fragment. (3) Methylated Illumina Y-shaped adapters are ligated. (4) Bisulfite conversion opens the DNA structure revealing a single-stranded DNA molecule that can be amplified using the Illumina adapters. Sequencing requires paired-end reads to obtain both the methylation and the genomic sequence information. For more details on the experimental procedure, see Supplemental Figure 1A and Supplemental Protocol . ( B ) Deconvolution procedure: for more details on the bioinformatics analysis, see Supplemental Figure 1B .

Article Snippet: We used 100 ng of C. acetobutylicum genomic DNA to prepare an EM-seq library (NEB E7120) as directed by the manufacturer.

Techniques: Nick Translation, Synthesized, Methylation, Amplification, Sequencing

Journal: Genome Research

Article Title: Methyl-SNP-seq reveals dual readouts of methylome and variome at molecule resolution while enabling target enrichment

doi: 10.1101/gr.277080.122

Figure Lengend Snippet: Methyl-SNP-seq applied to bacteria. ( A ) Summary of genome assembly using deconvoluted reads. Genome coverage was measured against E. coli or E. coli + C. acetobutylicum reference genome using QUAST. ( B ) Hierarchy clustering of significantly enriched 8-mers and the corresponding motif logo (generated by WebLogo ) for E. coli and mixed sample ( E. coli + C. acetobutylicum ). The number at the bottom represents the number of enriched k -mers in each cluster. ( C ) Identified methyltransferase motif for the top 100 largest assembled contigs. Each dot represents a contig.

Article Snippet: We used 100 ng of C. acetobutylicum genomic DNA to prepare an EM-seq library (NEB E7120) as directed by the manufacturer.

Techniques: Generated

Journal: bioRxiv

Article Title: Base editing as a genetic treatment for spinal muscular atrophy

doi: 10.1101/2023.01.20.524978

Figure Lengend Snippet: a , Number of putative off-target sites in the human genome with up to 2 mismatches for each gRNA, annotated by CasOFFinder . Predictions for SpCas9 utilized an NGG, NAG, or NGA PAM; with SpRY, a PAMless NNN search. b , Total number of CHANGE-seq detected off-target sites, irrespective of assay sequencing depth. c , Number of CHANGE-seq identified off-target sites that account for greater than 1% of total CHANGE-seq reads and are common across all 5 SMA fibroblast lines. d , Percentage of CHANGE-seq reads detected at the on-target site relative to the total number of reads in each experiment. For panels b, c , and d , mean, s.e.m, and individual datapoints shown for n = 5 independent biological replicate CHANGE-seq experiments (performed using genomic DNA from each of the 5 SMA fibroblast lines). e , f , Summary of targeted sequencing results from ABE-edited SMA fibroblasts or HEK 293T cells at the top 34 CHANGE-seq nominated off-target sites (common sites across all 5 SMA fibroblast lines and treatments with SpRY or SpRY-HF1), analyzing statistically significant editing of any adenine in the target site ( panel e ) or of all adenines in positions 1-12 of each of the 34 target sites ( panel f ).

Article Snippet: Experiments were halted after 72 hours and genomic DNA (gDNA) was collected by discarding the media, resuspending the cells in 100 μL of quick lysis buffer (20 mM Hepes pH 7.5, 100 mM KCl, 5 mM MgCl 2 , 5% glycerol, 25 mM DTT, 0.1% Triton X-100, and 60 ng/μL Proteinase K (New England Biolabs; NEB)), heating the lysate for 6 minutes at 65 ºC, heating at 98 ºC for 2 minutes, and then storing at -20 ºC.

Techniques: Sequencing

Journal: bioRxiv

Article Title: Telomeric DNA breaks in human induced pluripotent stem cells trigger ATR-mediated arrest and telomerase-independent telomere length maintenance

doi: 10.1101/2023.01.19.524780

Figure Lengend Snippet: A) Heat map depicting fold change in relative expression normalized to unedited parental cells of HR-related genes throughout the 48-hour induction period. B) GSEA indicating a significant enrichment of the homology-direct repair reactome gene signature, but not the microhomology-mediated / alternative end-joining signature, at 48 hours of TRF1-FokI induction. C) Significant upregulation (measured by Log2(fold change) vs. unedited parental cells) of many genes associated with homologous recombination at 48 hours of TRF1-FokI induction. D) Chromosome orientation (CO)-FISH performed on metaphase spreads harvested from uninduced (left, dox only) and 4-hour induced (right) TRF1-FokI iPSCs. Leading strand telomeres were hybridized with a 488-conjugated TelG PNA FISH probe (green) and lagging strand telomeres were hybridized with a Cy3-conjugated TelC PNA FISH probe (red). Top panels: scale bar=10 μm. Enlarged panels: scale bar=2 μm. E) Quantification of T-SCE frequency as detected by CO-FISH. Data are representative of 3 independent experiments. F) Combined IF-FISH staining for telomeric DNA (green) and PML protein (red) in uninduced control (top panels) and 48-hour induced (bottom panels) TRF1-FokI iPSCs. Arrows indicate representative ALT-associated PML bodies (APBs). Scale bar=5 μm. G) Quantification of discrete number of APBs per nucleus in uninduced control and 48-hour induced TRF1-FokI iPSCs. Data are representative of at least 100 nuclei across 3 independent experiments. p<0.0001 from unpaired two-tailed t-test. H) Quantification of the fraction of cells counted containing one or more APBs in uninduced control and 48-hour induced TRF1-FokI iPSCs. Data are representative of at least 100 nuclei across 3 independent experiments. p=0.006 from unpaired two-tailed t-test. I) Representative dot blot from C-circle assay (CCA) performed using genomic DNA harvested from uninduced, 8-hour induced, and 24-hour induced TRF1-FokI iPSCs (N=2 biological replicates shown). ALT+ U2OS and ALT-HEK293T DNAs were included as positive and negative controls, respectively. CCA was performed on bulk genomic DNA (top) and following removal of linear DNA by exonuclease digestion (bottom) to confirm circularity of template. Sample-specific background is quantified from signal generated in the absence of ϕ29 polymerase. J) Quantification of relative C-circle abundance in uninduced (0 hr) and induced (8 hr, 24 hr) TRF1-FokI iPSCs with and without exonuclease treatment to remove linear genomic DNA. C-circle signal for each sample is normalized to sample-specific background. Data represent average and SD from 2 technical replicates and 2 biological replicates (N=4).

Article Snippet: To remove linear genomic DNA, DNA samples were digested with a mixture of CviAII, BfaI, MseI, and NdeI for 16 hours at 25°C followed by 8 hours at 37°C, then digested with 12.5 U λ Exonuclease (NEB) and 100 U Exonuclease I (NEB) for 2 hours at 37°C.

Techniques: Expressing, Homologous Recombination, Staining, Two Tailed Test, Dot Blot, Generated

Journal: bioRxiv

Article Title: Woronin bodies move dynamically and bidirectionally by hitchhiking on early endosomes in Aspergillus nidulans

doi: 10.1101/2023.01.20.524968

Figure Lengend Snippet: (A-B) Protein domain cartoons for A. nidulans DipA (A) and PxdA (B). The domains previously defined as CC1 and CC2-3 are indicated. Green bars represent coiled coils predicted in this study. (C) Phylogenetic tree showing conservation of DipA and PxdA across 33 species representing major fungal clades, indicated by gray and pink boxes (clade abbreviations: Saccharo. = Saccharomycotina; Taphrino = Taphrinomycotina; Puccin. = Pucciniomycotina; Usti. = Ustilagomycotina; Agarico. = Agaricomycotina; Muc. = Mucoromycotina; Chytrid. = Chytridiomycota). Dictyostelium discoideum was included as an outgroup. Protein domain cartoons are scaled to one another to display amino acid length and domain organization in different species.

Article Snippet: Briefly, fragments were amplified using PCR (NEB Q5 highfidelity DNA polymerase, M0491) from other plasmids or A. nidulans genomic DNA.

Techniques: