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