Journal: Cell Reports Methods

Article Title: MASTR-seq enables multiplexed analysis of short tandem repeats with sequencing

doi: 10.1016/j.crmeth.2026.101341

Figure Lengend Snippet: Schematic of MASTR-seq procedure Day 1: (Step 1) Samples are dissociated to single-cell suspension, and cells are lysed. RNA and protein are digested by RNase A and proteinase K, respectively. DNA is subsequently purified by commercial HMW gDNA extraction kit or phenol:chloroform:isoamyl alcohol (PCIA, 25:24:1)-based method. (Step 2) HMW genomic DNA quality control assays are performed using Nanodrop for DNA quality, Qubit for quantifying DNA concentration, and gel electrophoresis for DNA integrity. Day 2: crRNA/tracrRNA annealing and Cas9 RNP assembling are completed (not shown in this schematic) before (step 3) HMW DNA dephosphorylation, (step 4) Cas9-mediated RNP cleavage, and (step 5) dA-tailing. (Step 6) The quality of Cas9-mediated RNP cleavage efficiency is assessed by PCR amplification of the target region using flanking primers. (Step 7) Cas9-RNP-cleaved products are purified from salts using sodium acetate-ethanol precipitation. Days 2–3: (step 8) target DNA fragments are precisely size-selected using BluePippin. Day 4: (step 9) samples are barcoded in a PCR-free manner and pooled for multiplexing using nanopore native barcodes. (Step 10) Adapters are ligated onto the multiplexed DNA. Days 4–6: (step 11) DNA is loaded onto the Nanopore flow cell and sequenced for 48 h (Step 12) Raw data are processed by basecalling, aligning and demultiplexing sequentially using Dorado. Custom computational tools are employed for the analyses of single-allele DNA methylation and STR tract length. See also .

Article Snippet: In applications with 5 × 10 6 cultured cell numbers or 20 mg clinical sample, we can obtain HMW DNA using commercial kits (NEB #T3060S/3050S) and consistently achieve >20 μg high-quality HMW DNA ( ).

Techniques: Single Cell, Suspension, Purification, Extraction, Control, Concentration Assay, Nucleic Acid Electrophoresis, De-Phosphorylation Assay, Amplification, Ethanol Precipitation, Multiplexing, DNA Methylation Assay

Journal: Nature Communications

Article Title: Muscle-specific gene editing therapy via mammalian fusogen-directed virus-like particles

doi: 10.1038/s41467-025-64200-9

Figure Lengend Snippet: a Schematic diagram illustrating the production of MuVLP Cas9 . b The levels of muscular fusogens, and Cas9 in VLPs, MuVLPs, VLP Cas9 , and MuVLP Cas9 . c ELISA quantification of Cas9 molecules per MuVLP Cas9 . The values and error bars represent the means ± s.d. of n = 3 technical replicates. d Scheme of MuVLP Cas9 or other controls cocultured with six different cell lines for detecting the specificity of MuVLP Cas9 for gene editing. e Immunofluorescence visualization of the Cas9 protein in cells cocultured with VLP Cas9 or MuVLP Cas9 . Red: anti-Cas9 antibody, green: F-actin labeled with Alexa Fluor 488 phalloidin, blue: nuclei labeled with DAPI. Scale bar, 20 μm. f Detection of exon 4 excision by genomic PCR in iC2C12 cells transfected with MuVLP Cas9 or other controls. The unedited genomic product is 883 bp long, and the gene-edited product (asterisk) is 445 bp long. g Sanger sequencing of amplicons confirmed the deletion of exon 4 and the generation of a fused intron 3/4 in genomic DNA from iC2C12 cells transfected with MuVLP Cas9 . The data are presented as the means ± s.d. of n = 3 biological replicates ( c ) or are representative of three independent experiments ( b , e – g ). Source data are provided as a Source Data file.

Article Snippet: Genomic DNA was extracted from tissues and cultured cells using a Genomic DNA Extraction Kit (D0063, Beyotime) according to the manufacturer’s instructions.

Techniques: Enzyme-linked Immunosorbent Assay, Immunofluorescence, Labeling, Transfection, Sequencing