Journal: bioRxiv

Article Title: Functional Characterization of a Novel Long Non-Coding RNA in Leishmania braziliensis Identified Through Computational Screening for Conserved RNA Structures

doi: 10.1101/2025.01.14.632970

Figure Lengend Snippet: Characterization of LbrM2903_26_lncRNA45 (lncRNA45). (A) IGV representation of genomic regions on chromosome 26 of L. braziliensis MHOMBR75M2903, showing lncRNA44 and lncRNA45 (magenta), two coding sequences (CDSs) - hypothetical protein LBRM2903_260014500.1 and NMD3 family protein LBRM2903_260014600.1 (purple), and the predicted locus 709 (blue). (B) RNA-seq coverage (red stacking bars) of lncRNA45 (magenta) in axenic amastigotes (AXA), metacyclic promastigotes (META), and procyclic promastigotes (PRO). The target locus 709 is indicated in blue. (C) Detection of lncRNA45 as a transcript smaller than 500 bp in total RNA extract from L. braziliensis M2903 procyclic promastigotes, by northern blotting using a radiolabeled specific probe. rRNA served as a loading control. (D) Characterization of lncRNA45 size and processing pathways using RNA circularization. Total RNA from L. braziliensis M2903 promastigotes was extracted and treated with tobacco acidic phosphatase for decapping (TAP+) or left untreated (TAP-), followed by circularization with T4 RNA ligase. Complementary circular DNA (ccDNA) was synthesized by reverse transcription using a primer binding 200 nt from the 5’ end of lncRNA45. PCR amplification with primers lncRNA45_cF1 and lncRNA45_cR1 (shown in gray) was performed using ccDNA as a template, and PCR products were cloned into pGEM-T for sequencing (Hang, Deng et al., 2015). Sequences from three cDNA clones derived from TAP- (dark green) and four from TAP+ (light green) were analyzed. Poly(A) tails (red dots) were detected in 3 out of 7 transcript profiles, all derived from TAP+ templates. The most representative transcript profile among the clones is indicated by a black arrow.

Article Snippet: The reactions were incubated for 2 minutes on ice and after that 30 unities (U) of T4 RNA ligase (NEB) enzyme were added to the system together with 1x T4 Buffer, 1mM ATP and 40U of RNAse inhibitor RNasin® (Promega) in a final volume of 50µL.

Techniques: RNA Sequencing Assay, Northern Blot, Control, Synthesized, Reverse Transcription, Binding Assay, Amplification, Clone Assay, Sequencing, Derivative Assay

Journal: bioRxiv

Article Title: A Novel Tiled-Amplicon Sequencing Assay Targeting the Tomato Brown Rugose Fruit Virus ( ToBRFV ) Genome Reveals Widespread Distribution in Municipal Wastewater Treatment Systems in the Province of Ontario, Canada

doi: 10.1101/2023.11.02.565359

Figure Lengend Snippet: Kraken2 classified read pair counts at the domain-level for (A) RNA shotgun library prepared samples, and (B) ToBRFV -Seq prepared samples . At the domain-level, categories included are viral, archaea, bacterial, eukaryotic, or other reads, where other includes unclassified reads, plasmids, adapters, linkers, and/or primers. (C) The percentage of reads represented by each domain for both prep methods. Samples A-E were used to calculate the average and the blank was excluded. (D&E) Percentage of Kraken2 classified ToBRFV (T), all other Tobamoviruses (OT), and the remainder of virus classified reads (RR) (F). Counts of Tobamovirus classified read pairs at the species-level (G). (A,B,D&E) Reads are represented on a log-scale to magnify and visualize low-abundance reads. Two-tailed tests were used to compare the mean read counts of RNA shotgun and ToBRFV -Seq classified reads, respectively. (D) RNA shotgun samples, p < 0.05, n = 5, df = 4, t-values, (T-OT) 3.569, (T-RR) −1.015, & (RR-OT) −1.181, critical t-value ±2.776. (E) ToBRFV -Seq samples, p < 0.05, n = 5, df = 4, t-values, (T-OT) 12.523, (T-RR) 12.523, & (RR-OT) 2.449, critical t-value ±2.776. Thus, there is a statistically significant difference between the number of ToBRFV and other Tobamovirus reads produced by RNA shotgun sequencing. Whereas, there is a statistically significant difference between the number of ToBRFV reads produced compared to both other Tobamovirus reads and the read remainder.

Article Snippet: RNA extracts from WWI were prepared for RNA sequencing using the NEB Ultra II RNA Sequencing kit ( New England Biolabs , E7770S), following manufacturer’s recommended protocol, section 5.

Techniques: Virus, Two Tailed Test, Produced, Shotgun Sequencing

Journal: bioRxiv

Article Title: Coordination of transcription-coupled repair and repair-independent release of stalled RNA polymerase II in response to transcription-blocking lesions

doi: 10.1101/2024.07.07.602436

Figure Lengend Snippet: a, Schematic representation of Damage-seq and PADD-seq. For Damage-seq, genomic DNA extracted from UV-irradiated cells are sonicated and ligated to the first adaptor, followed by immunoprecipitation with the lesion-specific antibody. The precise sites of the lesions are determined by the stalling of primer extension with high-fidelity DNA polymerase. After the ligation of the second adaptor, the primer extension products are PCR-amplified and sequenced. For PADD-seq, PolII-associated DNA are enriched by regular chromatin- immunoprecipitation (ChIP) with an anti-pan-RPB1 antibody. Purified DNA fragments are subjected to Damage-seq. b, Diagram of the experimental design for the measurement of TCR (by Damage-seq) and PolII-CPD interaction (by PADD-seq). c, Meta-gene analysis of PADD-seq signals around TSSs and TESs for active genes longer than 50 kb ( n = 2790) under indicated conditions. d, Quantification of PolII retention on damage sites by relative change of PADD-seq signals from 0.5 h to 2 h on each gene. Active genes longer than 20 kb were selected ( n = 4488). e, Meta-gene analysis of Damage-seq signals around TSSs for active genes longer than 50 kb ( n = 2790) under indicated conditions. Cells were collected immediately (0 h) or at 8 h after UV irradiation. Fraction CPDs remaining was calculated as the ratio of 8 h to 0 h. f, Violin plots of relative Damage-seq signals on each active gene ( n = 6406). Log2 value of the ratio of fraction CPDs remaining on TS to that on NTS was calculated. P value was calculated using two-tailed paired Student’s t -test for (d) and (f) . See also Supplementary Fig. 1 and 2. Source data are provided as a Source Data file.

Article Snippet: In brief, 167 μg of fragmented chromatin and 25 μg of anti-RPB1 antibody (Bethyl Laboratories, A304-405A) were incubated in RIPA buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA, 140 mM NaCl, 1% Triton X-100, 0.1% SDS and 0.1% Na-DOC) supplemented with protease inhibitors, 0.1 % BSA (Sigma) and 0.1 μg/μl tRNA (Sigma) for 2 h on a rotator at 4 °C.

Techniques: Irradiation, Sonication, Immunoprecipitation, Ligation, Amplification, Chromatin Immunoprecipitation, Purification, Two Tailed Test

Journal: bioRxiv

Article Title: Coordination of transcription-coupled repair and repair-independent release of stalled RNA polymerase II in response to transcription-blocking lesions

doi: 10.1101/2024.07.07.602436

Figure Lengend Snippet: a, Meta-gene analysis of Damage-seq signals around TSSs for active genes longer than 50 kb ( n = 2790) under indicated conditions. Cells were collected immediately (0 h) or at 8 h after UV irradiation. Fraction CPDs remaining was calculated as the ratio of 8 h to 0 h. Data of WT and CSB -KO cells are from our previous study . b, Violin plots of relative Damage-seq signals on each active gene ( n = 6406). Log2 value of the ratio of fraction CPDs remaining on TS to that on NTS was calculated. c, Meta-gene analysis of PADD-seq signals around TSSs and TESs for active genes longer than 50 kb ( n = 2790) under indicated conditions. d, Quantification of PolII retention on damage sites by relative change of PADD-seq signals from 0.5 h to 2 h on each gene. Active genes longer than 20 kb were selected ( n = 4488). P value was calculated using two-tailed paired Student’s t -test for (b) and (d) . e, Diagram showing that three possible fates of lesion-stalled PolII should co-exist in RPB1-K1268R cells. See also Supplementary Fig. 5. Source data are provided as a Source Data file.

Article Snippet: In brief, 167 μg of fragmented chromatin and 25 μg of anti-RPB1 antibody (Bethyl Laboratories, A304-405A) were incubated in RIPA buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA, 140 mM NaCl, 1% Triton X-100, 0.1% SDS and 0.1% Na-DOC) supplemented with protease inhibitors, 0.1 % BSA (Sigma) and 0.1 μg/μl tRNA (Sigma) for 2 h on a rotator at 4 °C.

Techniques: Irradiation, Two Tailed Test

Journal: bioRxiv

Article Title: Efficient Genome Editing with Chimeric Oligonucleotide-Directed Editing

doi: 10.1101/2024.07.09.602710

Figure Lengend Snippet: ( a ) Schematic of the T4 RNA Ligase I-mediated cpegRNA synthesis. ( b ) Representative of denaturing gel showing successful ligation of sgRNA and ssDNA oligo to generate cpegRNA that targets mCherry gene. ( c ) Visualization of HEK293T cells by fluorescence microscopy showing the mCherry activation by PE2 and engineered CODE-Bst variants with ligated cpegRNA. Cells were transfected with prime editors and CODEs 72 hours prior to imaging. ( d ) Quantification of mCherry activation in (c) via flow cytometry. ( e ) Schematic of the workflow for transfection of plasmid encoding human codon-optimized CODE and synthetic or ligated cpegRNA. Error bars represent ± SD, where n = 3 biological replicates.

Article Snippet: Ligations were prepared using T4 RNA Ligase I (New England Biolabs, Cat# M0204) as follows: 3.5 μL T4 RNA Ligase Buffer, 4.5 μL PEG8000, 2 μL T4 RNA Ligase I, 3 μL 10mM ATP, 2 μL 100uM sgRNA, 5 μL 100 mM 5’ phosphorylated ssDNA, 0.25 μL Murine RNase Inhibitor (New England Biolabs, Cat# M0314), and 14.75 μL water.

Techniques: Ligation, Fluorescence, Microscopy, Activation Assay, Transfection, Imaging, Flow Cytometry, Plasmid Preparation