Journal: STAR Protocols

Article Title: Protocol to track single-cell-derived clones using DNA barcoding combined with single-cell RNA sequencing

doi: 10.1016/j.xpro.2025.104229

Figure Lengend Snippet: Semi-random barcode design Schematic of semi-random barcode oligonucleotide design with restriction sites used for insertion and a barcode ID tag.

Article Snippet: Package lentiviruses with HEK293T cells and the barcode library plasmid DNA using the Lipofectamine 3000 kit (Invitrogen) as per manufacturer’s instructions. a.

Techniques:

Journal: bioRxiv

Article Title: Spatial 5mC-seq profiling of embryos and decidua after implantation in mammal

doi: 10.64898/2025.12.15.694289

Figure Lengend Snippet: a , Schematic designs of the three versions of microchannel chips used in this study. b , Genome coverage of SmC-seq at the pseudobulk level under three different HCl treatment conditions. “3× HCl” indicates treatment with 0.2 N HCl at room temperature for 5 min per round, repeated for three rounds; “1× HCl” indicates a single round of the same treatment; “No HCl” indicates no HCl treatment. “Rep” denotes replicate. For each of the six samples, 69,256,892 raw reads were used as input. Genome coverage was defined as the proportion of CpGs across the whole genome that were covered by sequencing reads. c , Fluorescent imaging measuring cross-channel diffusion distances and possible crosstalk between two neighboring channels by alternately flowing 5-Carboxyfluorescein (5-FAM, Green)-labeled Barcode A and Cyanines3 (Cy3, Red)-labeled Barcode A in adjacent channels. E5.5 embryonic tissue sections, with or without three-rounds of HCl treatment, were evaluated and compared. The microchannel chip used here consists of 10 μm width channels with 5 μm spacing. d , Quantification of diffusion distances with and without three-rounds of HCl treatment. Two-side t-test was used for statistical analysis.

Article Snippet: For each channel, 3 μL of RNA barcode buffer (1.8x T4 Ligase buffer, 30 U/μL T4 DNA Ligase, 0.75x NEB Buffer 3.1, 0.2% Triton-X100, 5 U/μL RNase Inhibitor (Enzymatics), and 0.1 U/μL SUPERase•InTM RNase Inhibitor) and 1 μL of RNA barcode A were added.

Techniques: Sequencing, Imaging, Diffusion-based Assay, Labeling

Journal: bioRxiv

Article Title: Plasticity of extrachromosomal DNA segregation during drug adaptation

doi: 10.64898/2025.12.09.693337

Figure Lengend Snippet: Schematic of the scDNA-seq experimental workflow. The target regions were amplified, followed by barcode assignment within the emulsions. The emulsions were broken, adapters were added, and libraries were prepared for NGS. b, Reference regions used for normalization are indicated by red arrows. One or two reference regions were designed for each chromosome. c, Schematic of the two types of celularl barcodes, PseuMO-Tag and ID vector.

Article Snippet: For insertion, 2 μL of 100 μM barcode oligonucleotide and 1 μL of 100 μM reverse primer (5′-TGCAGCATGCGTCTCACAACG-3′) were mixed with 25 μL of NEBNext High-Fidelity 2× PCR Master Mix (New England Biolabs) and 22 μL of water to produce double-stranded barcode DNA (Thermo Fisher Scientific).

Techniques: Amplification, Plasmid Preparation

Journal: bioRxiv

Article Title: Plasticity of extrachromosomal DNA segregation during drug adaptation

doi: 10.64898/2025.12.09.693337

Figure Lengend Snippet: Schematic of single-cell cloning with cellular barcode. After PseuMO-Tag lentiviral cellular barcodes were transduced into each cell line, single cells were isolated into 96-well plates. Each clone was cultured until it reached approximately 1–2 × 10⁵ cells and cryopreserved. Once a sufficient number of clones was established, pooled clones were analyzed using scDNA-seq. For COLO320DM, the same clonal pool was additionally analyzed with scRNA-seq, and each clone was cultured individually. After 18 additional cell divisions, scDNA-seq was performed. b, Gamma distribution fitting of copy-number distributions of MYC for each clone derived from COLO320DM, H2170, and PC-3 cells. The dataset includes 5,861 cells from 12 clones in COLO320DM; 7,118 cells from 21 clones in H2170; and 14,796 cells from 25 clones in PC-3 cells. c, Heatmap of eight parameters of MYC for each clone shown in b, sorted in descending order of the mean value. Each column was normalized by Z-score scaling. d, Scatter plot of the mean and SD values for MYC copy number showing differences among clones. e, Gamma distribution fitting of MYC copy-number distributions in each COLO320DM clone. Orange curves represent the distributions at the same time point as in Fig. 2b –d and Extended Data Fig. 2. Purple curves represent the distributions after 18 additional cell divisions. f, Density plot of MYC expression levels for each clone. g, Correlation of the mean of MYC copy number and Mean MYC expression levels (left), and SD of MYC copy number and SD of MYC expression levels (right). h, Correlation of the mean of MYC copy number and mean of MYC target expression levels.

Article Snippet: For insertion, 2 μL of 100 μM barcode oligonucleotide and 1 μL of 100 μM reverse primer (5′-TGCAGCATGCGTCTCACAACG-3′) were mixed with 25 μL of NEBNext High-Fidelity 2× PCR Master Mix (New England Biolabs) and 22 μL of water to produce double-stranded barcode DNA (Thermo Fisher Scientific).

Techniques: Cloning, Isolation, Cell Culture, Clone Assay, Derivative Assay, Expressing