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: Scientific Reports

Article Title: Oxidative stress imbalance and cellular damage mediated by the ND4 G11778A mutation

doi: 10.1038/s41598-026-40061-0

Figure Lengend Snippet: Mitochondrial respiration in 661 W cells. The cells expressing different exogenous ND4 (with mutant ND4 gene or with wild-type ND4 gene) were cultured in medium with high glucose or galactose. ( A and B ) representative Oxygen Consumption Rates (OCR) profiles; ( C ) the basal OCR; ( P = 0.0005, P = 0.0258, n = 5 per group); ( D ) mitochondrial ATP production in the different groups( P = 0.039, n = 5 per group); ( E ) maximal OCR ( P <0.0001, P = 0.0038, n = 5 per group) ; ( F ) space capacity for mitochondria ( P = 0.0096, n = 5 per group). Data are presented as means ± SEM, n = 5, one-way ANOVA, * P < 0.05, ** P < 0.01, *** P < 0.001; **** P < 0.0001.

Article Snippet: Dulbecco’s modified Eagle’s medium (DMEM, high glucose, BIOEXPLORER, Cat#B1101-001); DMEM glucose-free medium (Basalmedia, Cat#L160KJ); Fetal bovine serum (FBS, Capricorn, Cat#F12A); Penicillin and streptomycin (Gibco, Cat#15140122); Galactose (Sigma-Aldrich, Cat#G5388); Puromycin (Basalmedia, Cat#S250J0); Cell-counting kit 8 (CCK-8) (Biosharp, Cat#BS350A); ROS assay kit (Solarbio, Beijing, China, Cat#CA1410); Superoxide Dismutase Assay Kit with WST-8 (Beyotime, Cat#S0101S); Catalase (CAT); Assay Kit (Solarbio, Cat#BC0205); GSH and GSSG Assay Kit (Beyotime, Cat#S0053); One-step TUNEL Apoptosis Kit (Elabscience, Cat#E-CK-A322); DAPI (Beyotime, Cat#C1005); Hoechst 33,342 (Solarbio, Cat#C0030); Seahorse XFe24 microplates (Agilent, Cat#02340); Agilent Seahorse XF DMEM medium (Agilent, Cat#103015); Sodium pyruvate (Agilent, Cat#103578); Glutamine (Agilent, Cat#103579); D-glucose (Agilent, Cat#103577); BCA protein assay kit (Beyotime, Cat#P0010); Mitochondrial Extraction Kit ( solarbio, Cat: SM0020); Blue/Clear Native-PAGE Gel Quick Preparation Kit (Meilunbio, MA0470); Acetylcysteine ( MedChemExpress, Cat#HY-B0215 ).

Techniques: Expressing, Mutagenesis, Cell Culture

Journal: Scientific Reports

Article Title: Oxidative stress imbalance and cellular damage mediated by the ND4 G11778A mutation

doi: 10.1038/s41598-026-40061-0

Figure Lengend Snippet: Mut-ND4 (m.G11778A) contributes to an oxidative stress imbalance and cellular damage. The upper panel illustrates cellular damage in 661 W cells expressing ND4 R340H. The lower panel compares normal intracellular physiological processes (left) with the effects in cells harboring the ND4 R340H (right), where antioxidants such as CAT, SOD, and GSH, are reduced, and ROS levels are increased. The ND4 R340H mutation disrupts ETC function, resulting in impaired mitochondrial oxygen consumption and reduced oxidative phosphorylation.

Article Snippet: Dulbecco’s modified Eagle’s medium (DMEM, high glucose, BIOEXPLORER, Cat#B1101-001); DMEM glucose-free medium (Basalmedia, Cat#L160KJ); Fetal bovine serum (FBS, Capricorn, Cat#F12A); Penicillin and streptomycin (Gibco, Cat#15140122); Galactose (Sigma-Aldrich, Cat#G5388); Puromycin (Basalmedia, Cat#S250J0); Cell-counting kit 8 (CCK-8) (Biosharp, Cat#BS350A); ROS assay kit (Solarbio, Beijing, China, Cat#CA1410); Superoxide Dismutase Assay Kit with WST-8 (Beyotime, Cat#S0101S); Catalase (CAT); Assay Kit (Solarbio, Cat#BC0205); GSH and GSSG Assay Kit (Beyotime, Cat#S0053); One-step TUNEL Apoptosis Kit (Elabscience, Cat#E-CK-A322); DAPI (Beyotime, Cat#C1005); Hoechst 33,342 (Solarbio, Cat#C0030); Seahorse XFe24 microplates (Agilent, Cat#02340); Agilent Seahorse XF DMEM medium (Agilent, Cat#103015); Sodium pyruvate (Agilent, Cat#103578); Glutamine (Agilent, Cat#103579); D-glucose (Agilent, Cat#103577); BCA protein assay kit (Beyotime, Cat#P0010); Mitochondrial Extraction Kit ( solarbio, Cat: SM0020); Blue/Clear Native-PAGE Gel Quick Preparation Kit (Meilunbio, MA0470); Acetylcysteine ( MedChemExpress, Cat#HY-B0215 ).

Techniques: Expressing, Mutagenesis, Phospho-proteomics