Journal: bioRxiv

Article Title: High-efficiency genomic mapping of chromatin-associated targets with CUT&RUN

doi: 10.1101/2024.12.03.626419

Figure Lengend Snippet: (A) Schematic of a representative CUT&RUN experiment. Camera icons depict expected observations at key quality control (QC) steps. (1) The quality of monodispersed cells or nuclei is determined by Trypan Blue staining, where the dye is excluded by native ‘viable’ cells (K562 shown), but enters permeabilized cells or isolated nuclei (MCF7 shown) [NOTES 17-19] . (2) Cells or nuclei are immobilized to ConA magnetic beads. Efficient bead binding is confirmed by examination of bound and unbound fractions under a microscope (HEK293 shown). The bead slurry is then aliquoted to 8-strip tubes for CUT&RUN. If applicable for the reaction target, SNAP-CUTANA TM nucleosome spike-ins (EpiCypher) are added [NOTES 1, 8-10] . Target-specific antibody [NOTES 2-10] is added and reactions incubated overnight at 4°C. (3) pAG-MNase is added, and activated by further addition of Ca 2+ , leading to selective cleavage and diffusion of antibody-labelled chromatin fragments to the supernatant. Reactions are supplemented with exogenous E. coli spike-in DNA to monitor to control for input and support normalization after NGS [NOTES 40] . (4) Bead-bound cells are removed on a magnet and target-enriched DNA purified from the supernatant. If using SPRI beads, avoid over-drying (they should retain a damp dark brown color) before elution. (5) CUT&RUN DNA is quantified by fluorometric assays (e.g., Qubit TM , Invitrogen TM ) and used to prepare sequencing libraries. Purified libraries are examined on a TapeStation or Bioanalyzer (Agilent Technologies) to determine molarity and confirm enrichment of mononucleosome-sized fragments (∼300 bp) [NOTES 48-52] . (6) Pooled CUT&RUN libraries are sequenced (e.g., Illumina), aiming for 5-10 million total reads per reaction (assuming a human genome), and data analyzed [NOTES 53-59] . (B) Important considerations at each step to ensure a successful CUT&RUN experiment.

Article Snippet: Any reaction spiked with an EpiCypher SNAP-CUTANA nucleosome panel ( e.g. , K-MetStat, HA Tag, or DYKDDDDK Tag [NOTE 1] ) can be analyzed using pipeline below.

Techniques: Control, Staining, Isolation, Magnetic Beads, Binding Assay, Microscopy, Stripping Membranes, Incubation, Diffusion-based Assay, Purification, Sequencing

Journal: bioRxiv

Article Title: High-efficiency genomic mapping of chromatin-associated targets with CUT&RUN

doi: 10.1101/2024.12.03.626419

Figure Lengend Snippet: (A) Schematic for antibody testing. Parallel reactions with cells / nuclei include controls (e.g., IgG [Negative], anti-H3K4me3 [Positive] and anti-H3K27me3 [Positive]; each with SNAP-CUTANA K-MetStat spike-in nucleosome panel) and the antibodies under test (with appropriate SNAP-CUTANA spike-in if available). (B) For PTMs and epitope-tagged targets, antibody specificity is determined by relative recovery of on- vs. off-target DNA-barcoded spike-in nucleosomes (see 3.3 ). Representative data show pass/fail metrics for five anti-H3K36me3 tested with K-MetStat Panel (signal for each antibody by row; for each spike-in nucleosome by column). Heatmap data (key on right) reflects percentage cross-reactivity (>20% = fail) after normalization to on-target (set to 100). (C) Antibody efficiency in CUT&RUN is determined by peak recovery after cell dilution. When challenged by reduced cell numbers an efficient antibody (#1) retains peaks lost by a mediocre reagent (#2) (500 –> 50 k; both anti-H3K36me3 in K562 cells). (D) For targets without an available spike-in, multiple antibodies are compared to identify that with best balance of high yields, robust S:N and rational enrichment. In the example: of five anti-TP53/p53 tested, #1 best generates strong peak structures associated with active transcription (i.e., high correlation with H3K4me3 / low with H3K27me3). Each CUT&RUN reaction was performed with 500 k native K562 cells; RPKM normalized reads were group scaled and aligned to genome build hg38.

Article Snippet: Any reaction spiked with an EpiCypher SNAP-CUTANA nucleosome panel ( e.g. , K-MetStat, HA Tag, or DYKDDDDK Tag [NOTE 1] ) can be analyzed using pipeline below.

Techniques:

Journal: PLoS ONE

Article Title: A rapid, specific, extraction-less, and cost-effective RT-LAMP test for the detection of SARS-CoV-2 in clinical specimens

doi: 10.1371/journal.pone.0266703

Figure Lengend Snippet: Cross-reactivity/exclusivity wet testing of the Prime CovidDetect ™ rapid detection kit.

Article Snippet: M. pneumoniae , M129 , ZeptoMetrix , NATPPA-BIO , 0/3.

Techniques: Virus

Journal: bioRxiv

Article Title: A 50-gene high-risk profile predictive of COVID-19 and Idiopathic Pulmonary Fibrosis mortality originates from a genomic imbalance in monocyte and T-cell subsets that reverses in survivors with post-COVID-19 Interstitial Lung Disease

doi: 10.1101/2023.10.22.563156

Figure Lengend Snippet: A . Study design of the 50-gene signature and cytokine analysis in COVID-19 patients (Cohort 1). B. Heatmap of COVID-19 patients based on the 50-gene signature discriminates three risk groups (low, intermediate, and high) based on SAMS. Every column represents a patient, and every row represents a gene. Log-based two-color scale is adjacent to the heatmap. Red denotes increased expression and green denotes decreased expression. Gene expression data is represented as Log2 normalized expression values. C-D. Time to death and time to discharge by day 60 in hospitalized patients with COVID-19, respectively. E-H. Plasma cytokine concentrations (IL6, IP-10, SPP1 and TGFβ) in low, intermediate, and high-risk profile patients with COVID-19 at days 2, 6 and 13 post admission. The data is presented as an average of triplicate values ± SEM for each group. Two-way ANOVA test (GraphPad software) Tukey’s multiple comparisons were used; * p<0.05. I. Study design of 7-gene signature analysis by RT-qPCR in PBMCs from COVID-19 patients (Cohort 2). J. Heatmap of COVID-19 patients based on the 50-gene signature discriminates three risk groups (low, intermediate, and high) based on SAMS Up scores. Heatmap nomenclature is the same as in . K-L. Time to death and time to discharge by day 60 in hospitalized patients with COVID-19 respectively in cohort two. The data is presented as an average of triplicated TUs values ± SEM for each group. * p<0.05

Article Snippet: We measured cytokine concentrations of 121 plasma samples from COVID-19 patients from Cohort 1 using a customized, Bioplex 200 compatible, human cytokine panel including, IL6, IP10, SPP1 and TGFβ-1 (#FCSTM18-06, R&D Systems).

Techniques: Expressing, Software, Quantitative RT-PCR