Journal: bioRxiv

Article Title: Synthetic circRNAs employ IRES activity for translation in cells and in cell-free translation systems

doi: 10.64898/2026.03.28.715045

Figure Lengend Snippet: (A) Experimental outline of the in vitro generated circRNA-based reporter assay based on the mRuby3_circ_EGFP_ScaI_v3-4 plasmid (pKL480) for evaluation of IRES activity of different IRES inserts, including inverse sequences as controls for circRNA translation dependent on insert length and GC-content. Following DNA template synthesis (PCR amplification from plasmid backbone using poly(A)- and poly(T)-including primers), circRNA was generated by T7 in vitro transcription. Linear RNA species were removed by RNase R-treatment and column purification. Resulting circRNA was subsequently used for in-cell transfection or for in vitro translation systems. EGFP and 3xHA-Nluc reporter systems were used throughout the following analyses. The plasmid-encoded mRuby was not used. (B) Illustration of the circularization reaction mediated by group I dt introns leading to self-spliced circRNA. The observed sequence scar formed by the remaining parts of the td introns are highlighted in light and dark red. Remaining split introns were generated as side products and need to be removed. (C) Overview of the tested IRES sequences indicating length and viral or cellular origin of the IRESes. (D) Quality control of the generated EGFP reporter including circRNAs using 1% FA-agarose gel. Linear side products (upper band) disappear by RNase R digestion. Only the circular RNase R-resistant band remains (lower band). 500 ng total RNA was loaded per lane. The RiboRuler High Range RNA Ladder (Thermo, #SM1821) serves as a reference. (E) Quality control of the generated 3xHA-Nluc encoding circRNAs using 1% FA-agarose gel. Linear side products (upper band) are mainly degraded by RNase R digestion. Only the circular RNase R-resistant band remains (lower band). Only Dlx1 shows less efficient circularization leading to a higher concentration of side products. Therefore, the RNase R incubation time was increased to 90 min. 500 ng total RNA was loaded per lane. RNA species: linear (L), circular (C), introns (I). (F) Quality control, using the High Sensitivity RNA ScreenTape, of the generated circRNA before purification (upper panel) and after RNase R digestion (lower panel). Remaining contaminants can be observed as light grey bands.

Article Snippet: The PCR products were purified using the Monarch PCR & DNA Cleanup kit (NEB, T1030L) and 1 μg DNA was subsequently used for in vitro transcription using the High Scribe T7 High Yield RNA synthesis Kit (NEB, E2040S) according to manufacturer’s protocol (20 μL total reaction volume) supplemented with 40 U RiboLock RNase inhibitor (Thermo, EO0381) and incubated for 2 hrs at 37°C and 800 rpm.

Techniques: In Vitro, Generated, Reporter Assay, Plasmid Preparation, Activity Assay, Amplification, Purification, Transfection, Sequencing, Control, Agarose Gel Electrophoresis, Concentration Assay, Incubation

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