Journal: Nature Communications

Article Title: Signal-noise metrics for RNA binding protein identification reveal broad spectrum protein-RNA interaction frequencies and dynamics

doi: 10.1038/s41467-023-41284-9

Figure Lengend Snippet: AGPC acidic guanidinium thiocyanate–phenol–chloroform, SRA SRA analysis. a Schematic illustration of the experimental approach. Repeated AGPC extraction enriches RBPs over non-RBPs; effect of repeated AGPC extraction on S / N was tested. Graphical representation of SRA: RNase-treated samples are compared to equivalent amounts of untreated samples by SDS-PAGE. RNA-bound proteins in untreated samples migrate at a higher molecular weight than their unbound counterparts. RNase digestion liberates RNA-bound RBPs allowing their mobilization into the separating gel. b Comparison of AGPC interphase samples isolated by methanol precipitation (95% v/v) following 1–6 AGPC extraction(s) of UV-crosslinked or non-crosslinked HeLa cells by SRA and Coomassie Blue (protein), SYBR Safe (RNA&DNA) staining; parallel gels. Protein (BCA) and RNA&DNA (UV-spectrophotometry) yields shown in the adjoining bar charts represent the mean ± 1 SD of three biologically independent samples, pooled (equivalent % fraction) for SRA analysis. c Immunoblot analysis of samples from ( b ). Sample compositions, target protein RBP–RNA interactome category, and target protein GO:RBP-annotation status indicated in the figure panel. Full bots are provided in Supplementary Fig. , RNA-binding domains and list of studies reporting UV-enrichment* provided in Supplementary Data . d Schematic illustration of enhanced S / N output. SRA is unable to distinguish non-RBPs from RBPs with low S / N in RNP fractions isolated by methanol precipitation (95% v/v) from the final AGPC interphase. SRA supports the identification of RNA enrichment methods that further enhance S / N , evidenced by a marked decrease in RNase-insensitive protein. Graphics ( a , b ) were created with BioRender.com. Experiments ( b , c ) were performed four times with similar results. Source data are provided as a Source Data file.

Article Snippet: Guanidinium thiocyanate (GT) buffer (4 M GT, 25 mM sodium citrate pH 7.0, 0.5% N-lauryl sarcosine, 5 mM EDTA pH 8.0, and 0.1 M 2-mercaptoethanol) was prepared with the following stock solutions prepared in DEPC-treated DI water: 5 M guanidinium thiocyanate (00522, Chemimpex), 750 mM sodium citrate pH 7.0 (BDH-9288, VWR; C-0759, Sigma), 10% N-lauryl sarcosine (L9150, Sigma), 0.5 M EDTA pH 8.0 (0105, VWR).

Techniques: Extraction, SDS Page, Molecular Weight, Comparison, Isolation, Staining, Spectrophotometry, Western Blot, RNA Binding Assay

Journal: Nature Communications

Article Title: Signal-noise metrics for RNA binding protein identification reveal broad spectrum protein-RNA interaction frequencies and dynamics

doi: 10.1038/s41467-023-41284-9

Figure Lengend Snippet: AGPC acidic guanidinium thiocyanate–phenol–chloroform, M methanol precipitation (95% v/v), I INP, L LEAP-RBP, SRA SRA analysis. a Comparison of RNP fractions isolated by M, I, and L methods from final AGPC interphase suspensions of UV-crosslinked cells by SRA and Coomassie Blue (protein), SYBR Safe (RNA&DNA) staining. Protein (BCA) and RNA (UV-spectrophotometry) yields shown in the adjoining bar charts represent the mean ± 1 SD of three biologically independent samples, pooled (equivalent % fraction) for SRA analysis. Effect of isolation method on protein yield analyzed using one-way ANOVA (Fisher’s PLSD post hoc test, unpaired, two-tailed, homoscedastic, no correction): significant main effect of isolation method (m*), F (2, 6) = 41.91, P < 0.001 (MvI*, P = 0.018; IvL*, P = 0.001; MvL*, P < 0.001). Effect of isolation method on RNA yield analyzed using a one-way ANOVA (Fisher’s PLSD post hoc test, unpaired, two-tailed, homoscedastic, no correction): non-significant main effect of isolation method (n.s.), F (2, 6) = 0.04, P = 0.965 (post hoc testing not applicable). b Immunoblot analysis of samples from a , sample compositions indicated in figure panel. c Pictorial representation of LEAP-RBP method: (A) Addition of chloroform and vortexing. (B) Addition of precipitation solution (LiCl and isopropanol), inversion and incubation for 1 minute; repeated 5+ times. (C) Vortexing (D) Centrifugation and rinsing with 95% methanol (v/v). Experiments ( a , b ) were performed twice with similar results. Source data are provided as a Source Data file.

Article Snippet: Guanidinium thiocyanate (GT) buffer (4 M GT, 25 mM sodium citrate pH 7.0, 0.5% N-lauryl sarcosine, 5 mM EDTA pH 8.0, and 0.1 M 2-mercaptoethanol) was prepared with the following stock solutions prepared in DEPC-treated DI water: 5 M guanidinium thiocyanate (00522, Chemimpex), 750 mM sodium citrate pH 7.0 (BDH-9288, VWR; C-0759, Sigma), 10% N-lauryl sarcosine (L9150, Sigma), 0.5 M EDTA pH 8.0 (0105, VWR).

Techniques: Comparison, Isolation, Staining, Spectrophotometry, Two Tailed Test, Western Blot, Incubation, Centrifugation

Journal: Nature Communications

Article Title: Signal-noise metrics for RNA binding protein identification reveal broad spectrum protein-RNA interaction frequencies and dynamics

doi: 10.1038/s41467-023-41284-9

Figure Lengend Snippet: AGPC acidic guanidinium thiocyanate–phenol–chloroform, L LEAP-RBP. Sample preps: s.p.1, L; s.p.2, L w/ DNA depletion step; s.p.3, repeated AGPC extraction and L w/ DNA depletion step. a SRA analysis of L fractions isolated from AGP input suspensions containing equivalent amounts of UV-crosslinked or non-crosslinked cells by s.p.1, s.p.2, s.p.3; parallel gels; Coomassie Blue (protein), SYBR Safe (RNA&DNA). Protein (BCA) and RNA&DNA (UV-spectrophotometry) yields shown in the adjoining bar charts represent the mean ± 1 SD of three biologically independent samples, pooled (equivalent % fraction) for SRA analysis. Effect of UV cross-linking and sample prep on protein yield of L analyzed using two-way ANOVA: significant interaction, F (2, 12) = 4.61, P = 0.033. Subdivided by largest main effect (m.e.), UV cross-linking (m.e.1*, dashed line), F (1, 12) = 93.66, P < 0.001. Effect of sample prep on protein yield of L for UV-crosslinked and non-crosslinked subgroups analyzed using independent one-way ANOVAs (Fisher’s PLSD post hoc test, unpaired, two-tailed, homoscedastic, no correction): non-crosslinked, significant m.e. of sample prep (m.e.2*), F (2, 6) = 6.07, P = 0.036 (e.1*, P = 0.023; e.2*, P = 0.023; n.s.1, non-significant, P = 0.998); UV-crosslinked, significant m.e. of sample prep (m.e.3*), F (2, 6) = 6.86, P = 0.028 (e.3*, P = 0.010; n.s.2, non-significant, P = 0.076; n.s.3, non-significant, P = 0.173). Effect of UV cross-linking and sample prep on RNA&DNA yield of L analyzed using two-way ANOVA: significant interaction, F (2, 12) = 719.45, P < 0.001. Subdivided by largest m.e., sample prep (m.e.4*, dashed lines), F (1, 12) = 998.29, P < 0.001. Effect of UV cross-linking on RNA&DNA yield of L for s.p.1, s.p.2, s.p.3 subgroups analyzed using independent one-way ANOVAs (Fisher’s PLSD post hoc test, unpaired, two-tailed, homoscedastic, no correction): s.p.1, non-significant m.e. of UV cross-linking (n.s.4), F (1, 4) = 0.14, P = 0.729; s.p.2, non-significant m.e. of UV cross-linking (n.s.5), F (1, 4) = 0.91, P = 0.393; s.p.3, significant m.e. of UV cross-linking (m.e.5*), F (1, 4) = 746.88, P < 0.001. b Immunoblot analysis of samples from ( a ), sample compositions indicated in figure panel. Experiments ( a , b ) were performed four times with similar results. Source data are provided as a Source Data file.

Article Snippet: Guanidinium thiocyanate (GT) buffer (4 M GT, 25 mM sodium citrate pH 7.0, 0.5% N-lauryl sarcosine, 5 mM EDTA pH 8.0, and 0.1 M 2-mercaptoethanol) was prepared with the following stock solutions prepared in DEPC-treated DI water: 5 M guanidinium thiocyanate (00522, Chemimpex), 750 mM sodium citrate pH 7.0 (BDH-9288, VWR; C-0759, Sigma), 10% N-lauryl sarcosine (L9150, Sigma), 0.5 M EDTA pH 8.0 (0105, VWR).

Techniques: Extraction, Isolation, Spectrophotometry, Sample Prep, Two Tailed Test, Western Blot

Journal: Nature Communications

Article Title: Signal-noise metrics for RNA binding protein identification reveal broad spectrum protein-RNA interaction frequencies and dynamics

doi: 10.1038/s41467-023-41284-9

Figure Lengend Snippet: AGPC acidic guanidinium thiocyanate–phenol-chloroform, I INP, L LEAP-RBP, SILAC SILAC LC–MS/MS analysis, SRA SRA analysis, E* significantly UV-enriched*, NE not E*. a Schematic illustration of the experimental approach. Heavy SILAC-labeled UV-crosslinked and light SILAC-labeled non-crosslinked cells were mixed prior to repeated AGPC extraction and isolation of RNP fractions by L or I methods. Graphic prepared in BioRender. b Volcano plots showing proteins identified as E* (red) or NE (blue) in I or L fractions by SILAC. Log 2 (CL/nCL) ratios were generated with SPI CL values and average SPI nCL values. Tested against the null hypothesis that protein log 2 (CL/nCL) ratios ( n = 3 biologically independent samples) are equal to 0 using unpaired, upper-tailed, heteroscedastic t tests (RNA-binding proteins are expected to be recovered from UV-crosslinked cells in greater amounts). Correction for multiple hypothesis testing was performed using the Benjamini–Hochberg approach and a false-discovery rate of 5%. c GO-enrichment analysis of proteins identified as E* in I or L fractions by SILAC (Fisher’s Exact, two-tailed, correction for multiple hypothesis testing performed using the Benjamini–Hochberg approach and a false-discovery rate of 5%). d Venn diagram showing overlap of total and E* proteins identified in I and/or L fractions by SILAC. Pie charts showing the number of RBPs and non-RBPs identified as E* or NE in I or L fractions by SILAC. For information on protein ID assignment and protein lists for each category or GO term used in this study, see Supplementary Data and . e Stacked bar charts showing the number of proteins with annotated RNA-binding or RNA-related functions identified as E* or NE in I or L fractions by SILAC. f Histograms showing average log 2 (CL/nCL) ratios of RBPs and non-RBPs identified in I or L fractions by SILAC. Font color for individual proteins reflects conclusions from SRA and immunoblot analysis of total clRNP fractions; red text: RNase-sensitive RBP; blue text: undetected or RNase-insensitive protein. Asterisks: E*. I and L SILAC LC–MS/MS experiments ( a ) were performed once with three biologically independent samples for each SILAC label group (CL, nCL). Source data are provided as Source Data file.

Article Snippet: Guanidinium thiocyanate (GT) buffer (4 M GT, 25 mM sodium citrate pH 7.0, 0.5% N-lauryl sarcosine, 5 mM EDTA pH 8.0, and 0.1 M 2-mercaptoethanol) was prepared with the following stock solutions prepared in DEPC-treated DI water: 5 M guanidinium thiocyanate (00522, Chemimpex), 750 mM sodium citrate pH 7.0 (BDH-9288, VWR; C-0759, Sigma), 10% N-lauryl sarcosine (L9150, Sigma), 0.5 M EDTA pH 8.0 (0105, VWR).

Techniques: Liquid Chromatography with Mass Spectroscopy, Labeling, Extraction, Isolation, Generated, RNA Binding Assay, Two Tailed Test, Western Blot

Journal: Nature Communications

Article Title: Signal-noise metrics for RNA binding protein identification reveal broad spectrum protein-RNA interaction frequencies and dynamics

doi: 10.1038/s41467-023-41284-9

Figure Lengend Snippet: N/AGPC neutral/acidic guanidinium thiocyanate–phenol-chloroform, AGP acidic guanidinium thiocyanate–phenol, L LEAP-RBP, X XRNAX, O OOPs, P Ptex, T TRAPP, R RIC, SILAC, SILAC LC–MS/MS analysis, SRA SRA analysis, E* significantly UV-enriched*, NE not E*. a Experimental flow outlining the main steps of LEAP-RBP and five referenced RNA-centric methods. TBE gel analysis was performed on 1 µg of RNA isolated by NGPC extraction of proteinase K-treated RNP fractions isolated from UV-crosslinked cells and an equivalent % fraction of their corresponding non-crosslinked samples. UV-dependence of protein and RNA recovery as well as S / N were evaluated by SRA with SYBR Safe (RNA&DNA), Coomassie Blue (protein), and silver stain (RNA, DNA, and protein) staining ( b ) or immunoblot ( c ). Sample compositions and/or normalization values are indicated in figure panels b, c . Immunoblot targets found UV-enriched* in referenced studies (Supplementary Data ) were marked with gold asterisks in c ; black asterisk (T): no yeast homolog, hence n/a; n.d.: not detected. d Specificity and selectivity of the different methods for RNA-bound RBPs was evaluated by comparing observed abundances of RBPs and non-RBPs as a function of their log 2 ( S / N ) ratios (SILAC and non-SILAC). Differences in corresponding frequency curves plotted as a function of log 2 ( S / N ) (solid) or log 10 (%TP) (dashed) indicate differences in protein–RNA adduct enrichment efficiency ( S / N ) or abundance (%TP), respectively. e Stacked bar charts showing the number of RBPs and non-RBPs identified as E* or NE by each method, or the estimated %TP S and %TP N contributions of RBPs and non-RBPs in each RNP fraction. Experiments were performed once ( a – c ); n = 1 (X, O, P, T, R); n = 3 biologically independent samples (L), pooled (equivalent % fraction) for SRA. Input samples isolated from AGP input suspensions containing UV-crosslinked and/or non-crosslinked HeLa cells were used as inter-run controls during SRA analysis, n = 1. Complete MS datasets and parameters discussed in this study for each of the referenced studies are provided as individual Excel files (Supplementary Data 10–20). Source data are provided as a Source Data file.

Article Snippet: Guanidinium thiocyanate (GT) buffer (4 M GT, 25 mM sodium citrate pH 7.0, 0.5% N-lauryl sarcosine, 5 mM EDTA pH 8.0, and 0.1 M 2-mercaptoethanol) was prepared with the following stock solutions prepared in DEPC-treated DI water: 5 M guanidinium thiocyanate (00522, Chemimpex), 750 mM sodium citrate pH 7.0 (BDH-9288, VWR; C-0759, Sigma), 10% N-lauryl sarcosine (L9150, Sigma), 0.5 M EDTA pH 8.0 (0105, VWR).

Techniques: Liquid Chromatography with Mass Spectroscopy, Isolation, Extraction, Silver Staining, Staining, Western Blot

Journal: Materials Today. Advances

Article Title: SARS-CoV-2 in wastewater: From detection to evaluation

doi: 10.1016/j.mtadv.2022.100211

Figure Lengend Snippet: An analogy of a possible data set collected by wastewater epidemiology of SARS-CoV-2. Viral load from wastewater concentration is calculated based on information and techniques used in Refs. [ , , ]. The total concentration of viral RNA in wastewaters decreases with no new infections. An increase in total concentration of viral RNA in the wastewaters signify possible viral outbreak in community.

Article Snippet: Auerswald and colleagues performed chemical inactivation of SARS-CoV-2 by using AVL buffer including carrier RNA; AVL buffer, GeneReach sample (contains guanidinium thiocyanate (GITC) and t-Octylphenoxypolyethoxyethanol (Triton X-100)) and Formaldehyde in PBS [ ].

Techniques: Concentration Assay