Journal:

Article Title: Consequences of Fas-Mediated Human Dendritic Cell Apoptosis Induced by Measles Virus

doi:

Figure Lengend Snippet: Fas-dependent apoptosis of MV-infected DCs in MV DC-T cell cocultures. (A) At day 5, apoptosis was analyzed by FACS in cocultures of MV-infected DCs and syngeneic activated T cells. MHC-II-FITC–CD3-PE doubling staining confirmed the FSC/SSC gates for DCs and T cells. These gates were used to analyze the DiOC6-PI double staining in the presence of ZB4 blocking anti-Fas antibody or related isotype control. Apoptotic dead cells have decreased mitochondrial transmembrane potential and permeabilized membranes that render them DiOC6 negative and PI positive, respectively. In contrast, viable cells are DiOC6 positive and PI negative. Results are representative of three experiments; standard deviations were below 10%. (B) Immature DCs were not infected or MV infected and then cultured for 24 h. RNAs were extracted and used for RNase protection using the hAPO-3 probe kit and developed by a PhosphorImager system for 6 h. Local background has been subtracted from each signal. The highest value (1,000) was attributed to the highest signal; then the levels of mRNAs were quantified by densitometry and scanning comparison with control probes (GAPDH and L32). Data shown are representative of three experiments. (C) DCs were not infected (thick line) or MV infected (gray histogram), cultured for 3 days, stained with antibodies against Fas (UB2-FITC) or a control antibody, and analyzed by FACS. The expression of Fas protein on gated viable DCs is shown. Data shown are representative of three experiments; standard deviations were below 10%.

Article Snippet: In brief, RNA was hybridized overnight with the in vitro-translated 32 P-labeled probe (hAPO-3 kit; Pharmingen).

Techniques: Infection, Staining, Double Staining, Blocking Assay, Cell Culture, Expressing

Journal: Developmental cell

Article Title: Xist Repeats A and B account for two distinct phases of X-inactivation establishment

doi: 10.1016/j.devcel.2020.05.021

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Labeled oligo probe pool (1–5 nM for Xist RNA FISH, 100 nM for Atrx or Mecp2 nascent RNA FISH) was added to hybridization buffer containing 25% formamide, 2x SSC, 10% dextran sulfate, and nonspecific competitor (0.1 mg/mL mouse Cot-1 DNA [Thermo Fisher Scientific]).

Techniques: Recombinant, Isolation, Multiplex Assay, Software

Journal: Methods (San Diego, Calif.)

Article Title: Functional analyses of mammalian virus 5′UTR-derived, small RNAs that regulate virus translation

doi: 10.1016/j.ymeth.2020.02.008

Figure Lengend Snippet: Schematic diagrams/workflows of protein-IRES RNA interaction assays. (A) Streptavidin pull-down of protein-biotinylated RNA complexes. The biotinylated RNA in this example is the EV-A71 5′UTR. RNA not labeled with biotin serves as a negative control for the assay. The red box highlights SL-II from the IRES. For simplicity, proteins are shown only bound to SL-II. For the Western blots, the presence or absence of biotin in the RNAs are indicated by the plus and minus signs, respectively. The absence of a protein signal in the ‘minus’ lanes indicates that cellular proteins did not bind non-specifically to the paramagnetic particles, i.e., the detected proteins were purified via their association with RNA. (B) Immunoprecipitation of native RNP complexes from cell lysates. Native RNP complexes are incubated with an antibody directed against an RNA-binding protein of interest; non-immune antibody serves as a negative control. Dynabeads coupled to protein A permit magnetic purification of RNP–antibody–protein A-Dynabead complexes. Beads are washed and RNA is eluted and purified. Specific target RNAs associated with the protein of interest are detected by Northern blot or qRT-PCR.

Article Snippet: Magnetic Dynabeads coupled to protein A (Invitrogen) are added for 30 min at 4 °C; beads are removed with a magnet.

Techniques: Labeling, Negative Control, Western Blot, Purification, Immunoprecipitation, Incubation, RNA Binding Assay, Northern Blot, Quantitative RT-PCR

Journal: Methods (San Diego, Calif.)

Article Title: Functional analyses of mammalian virus 5′UTR-derived, small RNAs that regulate virus translation

doi: 10.1016/j.ymeth.2020.02.008

Figure Lengend Snippet:

Article Snippet: Magnetic Dynabeads coupled to protein A (Invitrogen) are added for 30 min at 4 °C; beads are removed with a magnet.

Techniques: Reporter Assay, Hybridization, Modification, Stripping Membranes, Western Blot, Protease Inhibitor, Purification, Transfection, TA Cloning

Journal: Molecular Psychiatry

Article Title: Inhibition of colony stimulating factor 1 receptor corrects maternal inflammation-induced microglial and synaptic dysfunction and behavioral abnormalities

doi: 10.1038/s41380-020-0671-2

Figure Lengend Snippet: a Gene ontology (GO) biological processes pathway analysis shows that MIA microglia increase synaptogenic functions while repopulated microglia recover homeostatic functions. Left (red): Top significantly enriched GO biological process terms increased by MIA and decreased by repopulation. Right (purple): Top significantly enriched GO biological process terms decreased by MIA and increased by repopulation. These GO findings were verified using GORILLA. b IPA of genes with differential expression in microglia between MIA versus Saline (RNA-seq data). Pathway analysis reveals MIA-induced upregulation of neuritogenic gene expression, specifically in developmental stages, based on activation z -score. Red denotes pathway activated in E17 MIA microglia. c Genes in “neuritogenesis/formation of cellular protrusions” function. Hierarchal clustering of gene sets based on relative expression values; red: high relative expression, blue: low relative expression. Cluster 1 represents genes increased in adult MIA microglia but reduced in MIA + MG-REP including Ctnnd2, Ncam2, and Ntrk2 . Cluster 2 represents genes increased in immature MIA microglia including Ncam2, Ntn, Ptn and Wnt5a . Cluster3 represents genes decreased in immature MIA microglia including Plau. In situ hybridization (ISH) and immunofluorescence of E17 Saline or MIA offspring in the cortical plate region. d mRNA of cellular protrusion/ neuritogenic genes ( Ctnnd2, Ncam2, Ntn, Ptn, and Wnt5a ) were detected by florescent-labeled antisense cRNA probes (red) but not by scramble cRNA probe (not detected: N.D.), and the sections were immunostained for IBA1 (green) and DAPI (blue). e The number of IBA1 + cells expressing the cellular protrusion/neuritogenic genes were quantified in the cortical plate region. n = (4–5/2) male mice/ litters per molecule for Saline and MIA, n = 3 for scramble control probe. * p < 0.05, ** p < 0.01, ns denotes no significance, by unpaired Student t test. Graphs indicate mean ± s.e.m. ELISA verification of selected RNA-seq molecules: CTNND2 ( f ), NCAM2 ( g ), NTRK2 ( h ), NTN ( i) , PTN ( j ) and WNT5A ( k ) in acutely isolated microglia. MIA increases protein expression of cellular protrusion/neuriotgenic molecules in microglia that were normalized via repopulation. n = (6/4, 6/3, 5/3, 6/3) female mice/litters for P60 Saline + CTRL, MIA + CTRL, Saline + MG-REP and MIA + MG-REP. CTNND2: Prenatal treatment effect, F (1,19) = 157.1, p < 0.0001, Drug effect, F (1,19) = 262.7, p < 0.0001, Interaction effect, F (1,19) = 201, p < 0.0001, NCAM2: Prenatal treatment effect, F (1,19) = 23.76, p = 0.0001, Drug effect, F (1,19) = 26.29, p < 0.0001, Interaction effect, F (1,19) = 17.63, p = 0.0005, NTRK2: Prenatal treatment effect, F (1,18) = 13.99, p = 0.0015, Drug effect, F (1,18) = 12.45, p = 0.0024, Interaction effect, F (1,18) = 0.06203, p = 0.8061, NTN: Prenatal treatment effect, F (1,19) = 0.01669, p = 0.8986, Drug effect, F (1,19) = 0.8, p = 0.3823, Interaction effect, F (1,19) = 6.121, p = 0.0230, PTN: Prenatal treatment effect, F (1,19) = 10.31, p = 0.0046, Drug effect, F (1,19) = 52.02, p < 0.0001, Interaction effect, F (1,19) = 0.002927 p = 0.9574, WNT5A: Prenatal treatment effect, F (1,19) = 5.581, p = 0.0290, Drug effect, F (1,19) = 1.550, p = 0.2282, Interaction effect, F (1,19) = 0.0834 p = 0.7799, * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001 as determined by 2-way ANOVA (alpha = 0.05) with Tukey’s post-hoc. # p < 0.05 for main effect of MIA. Graphs indicate mean ± s.e.m.

Article Snippet: For the detection of NCAM2, CTNND2 and WNT5A, custom ELISA kits were developed according to the manufacturer’s instruction using anti-NCAM2 goat polyclonal antibody (0.3 μg/well Acris Antibodies GmbH, AP32136PU-N), biotinylated anti-NCAM2 goat polyclonal antibody using Antibody Biotinylation Kit (0.3 μg/ml, Pierce/Thermo Scientific, 90407), anti-CTNND2 mouse monoclonal antibody (0.3 μg/well, Santa Cruz Biotechnology, SC-81793, clone 40.1), biotinylated anti-CTNND2 rabbit antibody (1 μg/ml, Abcam, EPR17628), anti-WNT5A goat polyclonal antibody (0.3 μg/well, R&D Systems, AF645), and biotinylated anti-WNT5A antibody using Antibody Biotinylation Kit (1 μg/ml, Pierce).

Techniques: Expressing, RNA Sequencing Assay, Activation Assay, In Situ Hybridization, Immunofluorescence, Labeling, Enzyme-linked Immunosorbent Assay, Isolation

Journal: BMC Biotechnology

Article Title: In situ detection of non-polyadenylated RNA molecules using Turtle Probes and target primed rolling circle PRINS

doi: 10.1186/1472-6750-7-69

Figure Lengend Snippet: Probe designs and methods for RNA detection . (A) A Padlock Probe contains two ends that are brought into close proximity through hybridization to a target RNA molecule (blue) after which they can be joined by a DNA ligase (the ligation site is indicated by an arrow). The probe also contains an intervening segment which does not hybridize to the target molecule, but completes the circle. This intervening segment can be rather freely designed in terms of length and sequence and is used as the probe identifier. Thus probes may be equipped with individual intervening segments for unique identification in multiplexed experiments. (B) A Turtle Probe consists of a target recognizing element and the identifier joined by a hairpin structure bringing the probe ends into close proximity on an internal ligation template (the ligation site is indicated with by arrow). (C) Illustration of the solid support setup where the capture oligonucleotide (purple) is covalently attached to the glass, the target RNA (blue) is hybridized to the capture oligonucleotide and the Turtle Probe (red) is hybridized to the 3'-end of the target RNA. (D-F) The target RNA (blue) provides a free 3'-end for the rolling circle reaction employing a ligated circle probe (red) (could be a Padlock- or Turtle Probe) as template for the localized DNA synthesis (grey polymerase forms black DNA). The rolling circle product, extending from the 3'-end of the target RNA, is then visualized with labeled oligonucleotide probes (green) recognizing the copies of the identifier element produced in the rolling circle reaction.

Article Snippet: Ligation was performed with 0.1 u/μL T4 DNA Ligase (Fermentas) in 1× T4 DNA ligation buffer (supplied with the T4 DNA ligase) supplemented with 1 u/μL Ribolock RNase Inhibitor (Fermentas) and 0.2 μg/μL BSA in a humidity chamber for 30 min at 37°C.

Techniques: RNA Detection, Hybridization, Ligation, Sequencing, DNA Synthesis, Labeling, Produced

Journal: Nature methods

Article Title: Streamlined and sensitive mono- and di-ribosome profiling in yeast and human cells

doi: 10.1038/s41592-023-02028-1

Figure Lengend Snippet: a, Schematic of library generation by OTTR or ligation-based protocols from a single pool of RNase I derived RPFs (green) from a sucrose cushion. The pool of RPFs were split unevenly after T4 PNK treatment with only 1:10th of the RPFs used in OTTR. In OTTR, each step before cDNA size selection occurs in 4 hours in a single tube. First, input RPF RNA was labeled by either ddA or ddG on the 3′ end before unincorporated ddRTPs were inactivated by rSAP. Lastly, two ordered jumps, the first initiated from the +1Y DNA/RNA primer duplex and the second initiated from a non-templated dG addition to the RPF cDNA to jump to the 3’C adapter template, yields a cDNA molecule with a 5’ and 3’ adapter flanking the complement of the RPF input. In ligation-based, the 3’ adapter is first adenylated on its 5’ end. THen, 3’ adapter ligation is carried out, followed by gel-based size selection and overnight elution. THe next day, material is precipitated and primer hybridization for reverse transcription occurs. Following reverse transcription, cDNA is purified by gel-base size-selection. After elution, cDNA is circularized. In these illustrations green/light green denoted the RPF sequence, orange/light orange denoted the R1 adapter sequence, blue/light blue denoted the R2 adapter sequence, gray/dark gray denoted the unique molecular identifier sequence, brown/light brown denoted the barcode sequence, red octagon denoted polymerase blocking groups, magenta triangle denoted a 3’ddR, and a magenta square denoted the dG non-templated addition. b, Comparison of gene-level ribosome occupancy estimates from libraries generated in (a). Read counts are for RPFs aligned to verified CDSs excluding those RPFs that are aligned to the first 15 and last 10 codons. Read counts for each gene were normalized by DESeq2. c, Comparison of mean codon-level occupancy estimates from libraries generated in (a). Aligned RPFs were assigned to an A-site codon and counted. These counts were then rescaled by the mean codon count for the gene, excluding those RPFs that are aligned to the first 15 and last 10 codons, and averaged across the translatome. d-e, Metagene averages around the start (left) and stop (right) codons for either (d) OTTR or (e) ligation-based libraries. Aligned RPFs for each CDS were first rescaled by the mean codon count for the gene, excluding those RPFs that are aligned to the first 15 and last 10 codons, and then averaged across the translatome. Footprints were tabulated according to either the 5′ aligned position alone (shown at top as a black line), or both 5′ aligned position and read length (shown at bottom as a matrix of distinct RPF lengths and positions). f, Per-codon contributions to iχnos machine learning models of RPF occupancy profiles. A model based on a widow of 13 codons (−7 to +5) around the A-site was compared with thirteen additional models, each omitting one codon from the model. The contribution of a codon position to RPF occupancy profile was inferred from the change in Pearson’s correlation coefficient between the predicted ribosome occupancy versus actual ribosome occupancy changed when the codon was omitted (Y-axis).

Article Snippet: Anecdotally, we advise RNase I RPFs be first subjected to Oligo Clean and Concentrate (Zymo Research) purification before T4-PNK dephosphorylation.

Techniques: Ligation, Derivative Assay, Selection, Labeling, Hybridization, Reverse Transcription, Purification, Sequencing, Blocking Assay, Comparison, Generated

Journal: Nature methods

Article Title: Streamlined and sensitive mono- and di-ribosome profiling in yeast and human cells

doi: 10.1038/s41592-023-02028-1

Figure Lengend Snippet: a, Size-selection of P1 nuclease RPF cDNA from OTTR by direct imaging of Cy5, the dye covalently linked to the 5′ end of the +1dY DNA/RNA adapter duplex primer (see Fig. 1a). The 30 nt and 40 nt RNA oligonucleotides used for RPF size selection (not shown) were also used in OTTR reactions parallel to those using input RPFs, to generate cDNA size-selection markers. Bromophenol blue formamide loading dye was used to resuspend OTTR cDNA for size selection to avoid xylene cyanol interference during Cy5 imagining. A 0.6X TBE 8% urea-PAGE was chosen for cDNA size selection since xylene cyanol and the no-insert OTTR adapter-dimer cDNA (~75 nt) co-migrate. For these reasons, xylene cyanol was included only in the peripheral lanes. Horizontal black lines indicate the boundaries for cDNA gel slice excision to remove adapter-dimer from desired cDNA library. All lanes are from the same gel. P1 RPF were either from sucrose cushion purified human 293T or sucrose cushion purified S288C yeast material after nuclease digestion. A 30 nt and 40 nt template control OTTR reaction was used to synthesize OTTR cDNA to enable cDNA size selection equivalent to RNA size selection. b, Read length distribution of yeast RNase I (blue) and P1 nuclease (red) RPFs from the CDS, excluding those sucrose cushion purified RPFs that are aligned to the first 15 and last 10 codons, as in Extended Data Fig. 1b. Counts were represented in RPM and averaged across replicates. c, Read length distribution of human RNase I (blue) and P1 nuclease (red) RPFs from the CDS, excluding those sucrose cushion purified RPFs that are aligned to the first 15 and last 10 codons, as in Extended Data Fig. 1b. Counts were represented in RPM and averaged across replicates d, Fraction of sucrose cushion purified RNase I RPF cDNA library sequencing reads mapped to each transcript class for yeast libraries generated by P1 nuclease or RNase I digestion. e, Fraction of sucrose cushion purified RNase I RPF cDNA library sequencing reads mapped to each transcript class for human libraries generated by P1 nuclease or RNase I digestion. f, Average per-base read coverage of cytosolic 18S and 25S or 28S rRNA from yeast (left) or human (right) ribosome profiles with P1 nuclease (red) or RNase I (blue). Coverage was represented in reads per million total reads, including reads mapping to rRNA, tRNA, ncRNA, mRNA, and other genomic loci) to emphasize relative proportion from the entire library. Material was purified from a sucrose cushion. g, As in (f) but for 5.8S and 5S rRNA coverage. h, As in (f) for mitochondrial rRNA coverage.

Article Snippet: Anecdotally, we advise RNase I RPFs be first subjected to Oligo Clean and Concentrate (Zymo Research) purification before T4-PNK dephosphorylation.

Techniques: Selection, Imaging, cDNA Library Assay, Purification, Control, Sequencing, Generated

Journal: Nature methods

Article Title: Streamlined and sensitive mono- and di-ribosome profiling in yeast and human cells

doi: 10.1038/s41592-023-02028-1

Figure Lengend Snippet: a, Fraction of RNase I RPF cDNA library sequencing reads mapped to each transcript class. Library generation artifacts included sequences that were adapter-only, shorter than 15 bases, or unmapped. b, Read length distribution of OTTR (blue) and ligation-based (red) RNase I RPFs from the CDS, excluding those RPFs that are aligned to the first 15 and last 10 codons. Counts were represented in RPM and averaged across replicates c, For each read length from 26 to 29 nt, the fraction of RPF alignments with mismatches at the 5′-most base of the alignment. For this analysis, alignments were permitted to only have a single mismatch to the reference. d, For each read length from 26 to 29 nt, the fraction of RPF alignments with an adenosine (A) at the 3′-most base of the alignment. For this analysis, alignments were permitted to only have a single mismatch to the reference. e, For each read length from 26 to 29 nt, the fraction of RPF alignments with a thymine (T) at the 3′-most base of the alignment. For this analysis, alignments were permitted to only have a single mismatch to the reference.

Article Snippet: Anecdotally, we advise RNase I RPFs be first subjected to Oligo Clean and Concentrate (Zymo Research) purification before T4-PNK dephosphorylation.

Techniques: Comparison, Ligation, cDNA Library Assay, Sequencing

Journal: Nature methods

Article Title: Streamlined and sensitive mono- and di-ribosome profiling in yeast and human cells

doi: 10.1038/s41592-023-02028-1

Figure Lengend Snippet: a, Metagene average profiles around the start (left) and stop (right) codons from sucrose cushion purified yeast RPFs generated by P1 nuclease (top, red) or RNase I (bottom, blue) digestion. The 5′ ends of aligned reads were counted, and counts for each gene were rescaled by the mean codon count for the gene, excluding those RPFs that are aligned to the first 15 and last 10 codons, prior to averaging. b, As in (a), for sucrose cushion purified human 293T cell RPFs generated by P1 nuclease (top, red) or RNase I (bottom, blue) digestion. c, Gene-level ribosome occupancy estimates from sucrose cushion purified yeast RPFs generated by P1 nuclease and RNase I digestion. Read counts for each gene were normalized by DESeq2. d, Gene-level estimates from sucrose cushion purified human 293T cell RPFs generated by P1 nuclease and RNase I digestion. Read counts for each gene were normalized by DESeq2. e, Average profile of yeast footprints at start codons for P1 nuclease (red, 30 – 40 nt) and RNase I (blue, 25 – 29 nt) libraries. Footprint alignments were counted separately for each gene monitoring read length as well as 5′ end position (left) and 3′ end position (right), then averaged as in (a). A heatmap shows footprint abundance according to length and end position (below), and the end position average summed across all lengths is shown (above each heatmap matrix. The 5′ and 3′ end averages are shown to the left and to the right, respectively, of a black vertical bar. A diagram of a translating ribosome footprint (top) indicates mRNA cleavage positions of P1 nuclease (red triangle) and RNase I (blue triangle). f, Average profile of human cell footprints at start codons for P1 nuclease (red, 33 – 40 nt) and RNase I (blue, 27 – 32 nt) libraries, as in (e). g-h, Comparison codon-level ribosome occupancy estimates from (g) yeast or (h) human cell RPFs generated by P1 nuclease and RNase I digestion, as in Fig. 1c. In (h), arginine codons are shown in red. i, Schematic of proposed P1 nuclease and RNase I cleavage sites around an mRNA-engaged ribosome. Increased frequency of an RPF terminal position is indicated by increasing color saturation.

Article Snippet: Anecdotally, we advise RNase I RPFs be first subjected to Oligo Clean and Concentrate (Zymo Research) purification before T4-PNK dephosphorylation.

Techniques: Purification, Generated, Comparison