licl buffer  (Thermo Fisher)

Journal: JCI Insight

Article Title: Extracellular matrix alterations in chronic ischemic cardiomyopathy revealed by quantitative proteomics

doi: 10.1172/jci.insight.196933

Figure Lengend Snippet: ( A ) Schematic representation of the workflow for MS-based proteomic analysis of protein extracts from human ICM and donor patient myocardial tissue. Soluble proteins are depleted in the first LiCl extraction (LiCl) followed by solubilization of “insoluble” ECM proteins by extraction with buffer containing the photocleavable surfactant, Azo. ( B ) CVs of protein intensities across extracts from failing ICM and nonfailing donor tissues. The median CV of protein intensities was below 5% for extracts from both groups, indicating high quantitative reproducibility. CVs were calculated by (SD/mean intensity) × 100% for each quantified value in each replicate. ( C ) Upset plot and bar graph (inset) showing more than 6,000 unique protein identifications overall, as well as the degree of overlap between groups. ( D and E ) Simplified depiction of major categories of ECM proteins (per MatrisomeDB, ref. ) ( D ) and breakdown of the number of proteins in each category identified in this study ( E ). The inner circle shows the total number of proteins in each category while the outer circle indicates those that were identified herein. Black segments correspond to proteins present in MatrisomeDB that were not identified in human ICM and donor myocardial tissue.

Article Snippet: Pellets were homogenized in 20 volumes (~300 μL) of LiCl extraction buffer (3 M LiCl, 1 mM TCEP, 10 mM EDTA, and 1× Halt Protease Inhibitor Cocktail from Thermo Fisher Scientific) using a handheld Teflon homogenizer (Bel-Art).

Techniques: Extraction

Journal: bioRxiv

Article Title: Contributions of Folded and Disordered Domains to RNA Binding by HNRNPR

doi: 10.1101/2025.05.01.651718

Figure Lengend Snippet: A) Bottom, schematic of HNRNPR and its respective domains: Full length (FL) (grey), RRMs (dark teal) and LCD (light purple). Below the schematic, RG (triangle) or RGG (diamond) repeats in HNRNPR are plotted. Top, disorder propensity plot (IUPRED2) of HNRNPR with ordered amino acids (black dot) and disordered amino acids (red dot) indicated. B) Schematic of RNA Bind-n-Seq (RBNS) workflow. C) Top, schematic of protein fragment used for RBNS. Bottom, ranked scatter plots of RBNS enrichments of all 6mers for HNRNPR FL. D) Fluorescence polarization (FP) binding curves (N=3) for HNRNPR FL incubated with AAAUU RNA (red dot), polyA RNA (grey triangle) and polyN RNA (black square). Data are mean ± standard deviation (SD). E) Top, schematic of protein fragment used for RBNS. Bottom, ranked scatter plots of RBNS enrichments of all 6mers for HNRNPR RRMs + . F) FP binding curves (N=3) for HNRNPR RRMs + incubated with AAAUU RNA (red dot), polyA RNA (grey triangle) and polyN RNA (black square). Data are mean ± SD. G) Top, schematic of protein fragment used for RBNS. Bottom, ranked scatter plots of RBNS enrichments of all 6mers for HNRNPR LCD. H) FP binding curves (N=3) for HNRNPR LCD incubated with AAAUU RNA (red dot), polyA RNA (grey triangle) and polyN RNA (black square). Data are mean ± SD.

Article Snippet: Following incubation and washing unbound proteins, we either incubated the RBP-beads complex with the stG4 pool in RBNS binding buffer (25 mM Tris-HCl, 150 mM KCl, 3 mM MgCl2, 500 mg/mL BSA, 20 units/mL SUPERase IN (Invitrogen, #AM2696)) or the stG4 pool with 7dG in RBNS LiCl binding buffer (25 mM Tris-HCl, 150 mM LiCl, 3 mM MgCl2, 500 mg/mL BSA, 20 units/mL SUPERase IN (Invitrogen, #AM2696)) for 1 hour at 4°C.

Techniques: Fluorescence, Binding Assay, Incubation, Standard Deviation

Journal: bioRxiv

Article Title: Contributions of Folded and Disordered Domains to RNA Binding by HNRNPR

doi: 10.1101/2025.05.01.651718

Figure Lengend Snippet: A) Schematic of an RNA G-quadruplex (rG4). B) Schematic of RBNS workflow with a modification: enrichments are calculated based on a curated list of 649 rG4 patterns. C) Ranked scatter plot for enrichments of rG4 patterns determined by RBNS for: FL (grey), RRMs + (dark teal) and LCD (light purple). Inset, number of rG4 patterns that have an enrichment value above 1 by RBNS for FL, RRMs + and LCD. Data are mean ± standard deviation (SD). D) FP binding curves (N=3) for FL, RRMs + and LCD incubated with (GGGA) 4 RNA. Data are mean ± SD.

Article Snippet: Following incubation and washing unbound proteins, we either incubated the RBP-beads complex with the stG4 pool in RBNS binding buffer (25 mM Tris-HCl, 150 mM KCl, 3 mM MgCl2, 500 mg/mL BSA, 20 units/mL SUPERase IN (Invitrogen, #AM2696)) or the stG4 pool with 7dG in RBNS LiCl binding buffer (25 mM Tris-HCl, 150 mM LiCl, 3 mM MgCl2, 500 mg/mL BSA, 20 units/mL SUPERase IN (Invitrogen, #AM2696)) for 1 hour at 4°C.

Techniques: Modification, Standard Deviation, Binding Assay, Incubation

Journal: bioRxiv

Article Title: Contributions of Folded and Disordered Domains to RNA Binding by HNRNPR

doi: 10.1101/2025.05.01.651718

Figure Lengend Snippet: A) Schematic of the stG4 pool with breakdown of different RNA sequences within the pool and applications of the pool adapted from . Comparison of log 2 RBNS enrichment for B) FL and C) LCD with guanine in KCl (y-axis) versus RT Stop Score of rG4s (x-axis), where an RT Stop Score of < -2 denotes folded rG4s. Colors represent the nucleotide composition in the rG4 loops. The minimum requirement for classification was to have the same nucleotide twice in adjacent positions or consecutives loops. D) Log 2 RBNS enrichment for LCD with 7dG rG4s (y-axis) versus log 2 RBNS enrichment for LCD with guanine rG4s in KCl (x-axis). Grey points represent non rG4s (RT Stop Score above -2) and gold points represent folded rG4s (RT Stop Score below -2). E) Boxplot shows the log 2 RBNS enrichment for LCD for non rG4 and folded rG4 with 7dG RNA (light green) and with guanine in KCl (light blue). Significance was determined by Wilcoxon test. Significance marks are as follows: **** (p ≤ 0.0001), ns (not significant). F) Cumulative distribution function (CDF) of log 2 RBNS enrichment for the LCD with guanine in KCl separated by the minimum G-tract allowed in an oligo. Inset shows p-values determined by a two-sided KS test corrected via the BH procedure. Red denotes significance and values are as follows: f (p≤0.0001). G) CDF of log 2 RBNS enrichment for LCD with guanine in KCl separated by minimum nucleotide counts allowed in the rG4 loops in an oligo. Inset shows p-values determined by a two-sided KS test corrected via the BH procedure. Red denotes significance and values are as follows: a (ns), d (p≤0.01), e (p≤0.001), f (p≤0.0001). H) Log 2 RBNS enrichment (y-axis) of specific examples of oligos bound by LCD with 7dG RNA (light green) and guanine in KCl (light blue). Bars represent the mean of enrichment and points denote individual enrichments.

Article Snippet: Following incubation and washing unbound proteins, we either incubated the RBP-beads complex with the stG4 pool in RBNS binding buffer (25 mM Tris-HCl, 150 mM KCl, 3 mM MgCl2, 500 mg/mL BSA, 20 units/mL SUPERase IN (Invitrogen, #AM2696)) or the stG4 pool with 7dG in RBNS LiCl binding buffer (25 mM Tris-HCl, 150 mM LiCl, 3 mM MgCl2, 500 mg/mL BSA, 20 units/mL SUPERase IN (Invitrogen, #AM2696)) for 1 hour at 4°C.

Techniques: Comparison

Journal: bioRxiv

Article Title: Massively parallel characterization of RNA G-quadruplex stability and molecular recognition

doi: 10.1101/2025.04.29.651304

Figure Lengend Snippet: A) Schematic of RNA G-quadruplex sequence features. The design of our structural rG4 pool focused on using the base rG4 sequence and varying the G-tract lengths, loop nucleotide composition, and loop length. This pool was in vitro transcribed with either guanine or 7-deaza-guanine (7dG), which is unable to fold into an rG4. B) Schematic of RT stop experimental workflow. C) Evaluation of RT stop protocol against a strong rG4 (r(AGGG) 4 AAAAAAA) either prepared with guanine or 7dG. Representative gel for RT stop products and full-length read through products and %RT Stop was quantified (n=2). D) Schematic of RT stop sequencing procedure and metric for evaluating RT Stop Scores. E) Bar graphs of a putative rG4 (G 3 AG 3 AG 3 AG 3 , red background) and a negative control (A 3 C 3 A 3 C 3 A 3 C 3 A 3 ) and their respective average frequencies in KCl (orange), LiCl (light orange), No Salt (white) and with 7dG (grey) (n=2). Cumulative distribution function (CDF) of F) RT Stop Scores at 40°C with different salt conditions (KCl in orange, LiCl in pink, and No Salt in black) and G) RT Stop Scores in KCl with different RT temperature conditions (30°C in light red, 40°C in red, and 50°C in dark red) (n=2). Plot insets ( left ) show p-values determined by two- sided KS test corrected by BH procedure. Red square indicates p≤0.05. Values are as follows: a (ns), d (p≤0.01), f (p≤0.0001). Zoomed in plots ( right ) shows RT Stop Scores less than 0.

Article Snippet: Beads with protein-RNA complexes were isolated in the magnetic stand (Invitrogen, 12321D) and thoroughly washed with either RBNS KCl wash buffer (25 mM Tris-HCl, 150 mM KCl, 20 units/mL SUPERase IN (Invitrogen, AM2696)) or with RBNS LiCl wash buffer (25 mM Tris-HCl, 150 mM LiCl, 20 units/mL SUPERase IN (Invitrogen, AM2696)).

Techniques: Sequencing, In Vitro, Negative Control

Journal: bioRxiv

Article Title: Massively parallel characterization of RNA G-quadruplex stability and molecular recognition

doi: 10.1101/2025.04.29.651304

Figure Lengend Snippet: A) Chemical structure of pyridostatin (PDS). B) Schematic of expected interaction and RT Stop consequence by PDS addition. C) Scatter plots of putative rG4 sequences by RT Stop Score in KCl and PDS (y-axis) v. RT Stop Score in KCl alone (x-axis) colored by minimum G-tract length (GG (purple), GGG (red), GGGG (yellow)) (Data is the average of 3 independent replicates). Dashed horizontal line shows PDS score of 0. D) Box plot of putative rG4s by PDS Score in KCl with total loop length. Colored by fixed G-tract length (GG (light orange), GGG (orange), GGGG (dark orange)) (Data is the average of 3 independent replicates). Dashed horizontal line shows PDS score of 0. E) Box plot of putative rG4s by PDS Score in LiCl (log 2 (Sequence Freq LiCl + PDS /Sequence Freq LiCl ) with total loop length. Colored by fixed G-tract length (GG (light orange), GGG (orange), GGGG (dark orange)) (Data is the average of 3 independent replicates). Dashed horizontal line shows PDS score of 0. F) Box plot of putative rG4s by PDS Score with different loop compositions (C (red), A (blue), U (gold)). Fixed G-tracts rG4s are shown as points colored by their G-tract length (GG (light orange), GGG (orange), GGGG (dark orange)). Dashed horizontal line shows PDS score of 0. Inset shows p-values determined by two-sided KS test corrected by BH procedure. Red square indicates p≤0.05. Values are as follows: d (p≤0.01), f (p≤0.0001). G) Bar graphs of putative rG4s with U loops and their respective average RT Stop Scores in KCl (red) and in KCl and PDS (purple) (n=3). Error bars (SD) generated from three independent replicates. Dashed line at -2 RT Stop Score is indicative for folded rG4s. Colored rectangles cluster sequences by observed trends by PDS-induced decreases in RT Stop Scores, either: no change (red), slight decrease (light orange), decrease (orange), and large decrease changing to ‘folded rG4’ (yellow). H) Ranked plot of U2AF2 mutation sequences which correlate sequence ΔWT Score (colors correspond to ΔWT Score (red is positive, destabilizing; grey is zero; blue is negative, stabilizing)) and PDS Score (orange) (PDS score plotted on same axis) (Data is the average of 3 independent replicates). Dashed horizontal line shows ΔWT Score and PDS Score of 0. I) Combined heatmap of the U2AF2 rG4 sequences by ΔWT PDS Score. Fill gradient shifts from red (positive, more readthrough in PDS), white (0, equal to WT), to blue (negative, less readthrough in PDS) were used (Data is the average of 3 independent replicates). Sequences were plotted by nucleotide or triplet mutated and full WT sequence for the natural rG4 is shown (x-axis, bottom heatmap). Triplets which contain GGG and their directly adjacent triplets ( top ) are colored in orange, while single G nucleotides ( bottom ) are colored in orange.

Article Snippet: Beads with protein-RNA complexes were isolated in the magnetic stand (Invitrogen, 12321D) and thoroughly washed with either RBNS KCl wash buffer (25 mM Tris-HCl, 150 mM KCl, 20 units/mL SUPERase IN (Invitrogen, AM2696)) or with RBNS LiCl wash buffer (25 mM Tris-HCl, 150 mM LiCl, 20 units/mL SUPERase IN (Invitrogen, AM2696)).

Techniques: Sequencing, Generated, Mutagenesis

Journal: bioRxiv

Article Title: Massively parallel characterization of RNA G-quadruplex stability and molecular recognition

doi: 10.1101/2025.04.29.651304

Figure Lengend Snippet: A) Schematic of G3BP1 and FMRP with their respective disorder propensity plot. B) Schematic of RNA bind-n-seq (RBNS). Correlation plots of the log 2 RBNS enrichment of C) G3BP1 (+ Guanine) (y- axis) and D) FMRP LCD (+ Guanine) (y-axis) versus RT Stop Score in KCl (x-axis). Pearson’s correlation coefficient and p-value are included. E) Log 2 RBNS enrichment (Data is the average of 2 independent replicates) of unique oligos bound by FMRP LCD (light blue) and G3BP1 (dark gold). Dark gold rectangles represent oligos in stG4 (+ Guanine) pool. Grey rectangles represent oligos in stG4 (+ 7dG) pool. CDF of log 2 RBNS enrichment of F) G3BP1 (+ Guanine) and G) FMRP LCD (+ Guanine) separated by the minimum G-tract allowed in an oligo. Inset shows p-values determined by two-sided KS test corrected by BH procedure. Red square indicates p≤0.05. Values are as follows: f (p≤0.0001). Scatter plots of the log 2 RBNS enrichment of FMRP LCD (y-axis) versus G3BP1 (x-axis) in H) (+ Guanine) and I) ( + 7dG). Colors represent combinations of the loop motifs of the putative rG4s. Heatmap of U2AF2 by ΔWT Score of J) G3BP1 and K) FMRP LCD. Gradient represents the log 2 RBNS enrichments for the change in WT sequence versus mutated sequence. Sequences were plotted by nucleotide or triplet mutated (for A only), and full WT sequence for the natural rG4 is shown (x-axis, bottom heatmap). Top heatmap (for A only) shows mutations of triplets to AAA and their impact, while the bottom heatmap shows single nucleotide mutations (U, C, A, G) at each position. Triplets which contain GGG and their directly adjacent triplets ( top ) are colored in orange, while single G nucleotides ( bottom ) are colored in orange.

Article Snippet: Beads with protein-RNA complexes were isolated in the magnetic stand (Invitrogen, 12321D) and thoroughly washed with either RBNS KCl wash buffer (25 mM Tris-HCl, 150 mM KCl, 20 units/mL SUPERase IN (Invitrogen, AM2696)) or with RBNS LiCl wash buffer (25 mM Tris-HCl, 150 mM LiCl, 20 units/mL SUPERase IN (Invitrogen, AM2696)).

Techniques: Sequencing