Journal: Nature chemistry

Article Title: Sequencing abasic sites in DNA at single-nucleotide resolution

doi: 10.1038/s41557-019-0279-9

Figure Lengend Snippet: Chemical tagging of DNA abasic sites. a ) Structures of naturally occurring aldehyde residues found in DNA. b ) Structure of HIPS probe 1 . c ) Stability of AP-ODN and probe 1 -labelled ODNs after biotinylation, to alkaline-cleavage (100 mM NaOH, 15 min). Reactions were carried out at 70°C except where labelled RT, where room temperature was used. 5-fC-1-biotin was obtained by extending the reaction time with 1 to 24 h. Reactions were followed by LC-MS. Results from three independent replicates are shown. d ) Workflow of chemical enrichment of abasic sites using probe 1 . Tagging of aldehyde residues followed by biotinylation through CuAAC and streptavidin pulldown enriches for all reacted aldehydes, whilst the removal of the biotin tag by alkaline-cleavage occurs only at AP sites. The truncated DNA fragments with 5’-phosphate termini are then recovered and prepared for next-generation sequencing. Sequencing reads stack up immediately after captured AP sites due to the site-specific DNA cleavage step via base-catalyzed elimination.

Article Snippet: Streptavidin pulldown: Magnesphere streptavidin beads (Promega, 50 μL) were pre-washed three times with 1X binding buffer (5 mM Tris pH 7.5, 0.5 mM EDTA, 1 M NaCl, 0.05% Tween 20) and resuspended in 50 μL of 2X binding buffer (10 mM Tris pH 7.5, 1 mM EDTA, 2 M NaCl, 0.1% Tween 20).

Techniques: Liquid Chromatography with Mass Spectroscopy, Next-Generation Sequencing, Sequencing

Journal: Nature chemistry

Article Title: Sequencing abasic sites in DNA at single-nucleotide resolution

doi: 10.1038/s41557-019-0279-9

Figure Lengend Snippet: Outline of snAP-seq and analysis of synthetic DNA spike-ins. a ) Enrichment of AP DNA relative to GCAT, 5-fC and 5-fU DNA, after first streptavidin enrichment. DNA was recovered under alkaline-cleavage conditions (100 mM NaOH, 70°C), before neutralization and purification. DNA was quantified by qPCR, and normalized to input DNA. The recovery of each DNA sequence was compared to AP DNA, and represented as a fold-enrichment of AP DNA. Apart from GCAT DNA, all primers were designed 3’- to each modification and therefore expected cleavage site to allow DNA amplification. Mean ± S.E.M. of three replicates are shown. b ) Recovery of DNA sequences after second streptavidin enrichment. AP DNA remains unbound and is recovered from the supernatant, whilst 5-fU DNA is captured on the beads. DNA was quantified by qPCR and normalized to the amount of DNA recovered after first streptavidin enrichment. Results from three replicates are plotted. c ) Library-preparation workflow of snAP-seq. d ) Distribution of reads aligning to each model DNA sequence after next-generation sequencing. The number of reads of each ODN was normalized to the corresponding reverse strand in the input sample (see ). Only the modification-containing, forward strands of each ODN are analyzed.

Article Snippet: Streptavidin pulldown: Magnesphere streptavidin beads (Promega, 50 μL) were pre-washed three times with 1X binding buffer (5 mM Tris pH 7.5, 0.5 mM EDTA, 1 M NaCl, 0.05% Tween 20) and resuspended in 50 μL of 2X binding buffer (10 mM Tris pH 7.5, 1 mM EDTA, 2 M NaCl, 0.1% Tween 20).

Techniques: Neutralization, Purification, Sequencing, Modification, DNA Amplification, Next-Generation Sequencing

Journal: Cell reports

Article Title: Differences in syncytia formation by SARS-CoV-2 variants modify host chromatin accessibility and cellular senescence via TP53

doi: 10.1016/j.celrep.2023.113478

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: 250 μg lysate was reduced with 5 mM DTT in 500 μL RIPA and then added to 60 μL Pierce streptavidin bead slurry (Thermo) equilibrated 2x with RIPA buffer.

Techniques: FLAG-tag, Luciferase, Virus, Recombinant, SYBR Green Assay, Activity Assay, Software, CRISPR