superase in  (Thermo Fisher)


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    Structured Review

    Thermo Fisher superase in
    SF1 structure and function in pre-mRNA splicing. ( A ) Schematic diagram of the early-stage SF1-U2AF 65 -U2AF 35 splicing-factor complex recognizing the 3′ splice site. ULMs are represented by “W.” White circles represent UHMK1-phosphorylation sites. BPS, branch point sequence; Py, polypyrimidine. ( B ) Domains of SF1, U2AF 65 , and U2AF 35 splicing factors. The boundaries of constructs used in this study are indicated by double-headed arrows. Zn, CCCH zinc knuckle; UHM, U2AF homology motif; ULM, U2AF ligand motif; RRM, RNA recognition motif, RS, arginine-serine repeat; KH-QUA2, K homology with quaking-2 motif; PA, protein A; circled P, UHMK1-phosphorylation sites. ( C ) Structure of the phosphorylated SF1 14–132 -U2AF 65 ). A line representing residues that are absent from the electron density connects the C-terminus of the ULM to the coiled-coil motifs. The tryptophan of the ULM (W22) and phosphorylated serines are shown as spheres and arginine residues that are mutated in this study are shown in stick representation. The inset shows a sequence alignment of the UHMK1-phosphorylated SPSP motif ( bold (between lines)). ( D and E ) Samples of ( D ) phospho-SF1 1–255 and ( E ) AdML L RNA under experimental conditions. Samples either before ( Pre- ) or after ( Post- ) incubating at 23°C for 2 h in the presence of a 10-fold excess of SF1 (U) or phospho-SF1 (P) were analyzed by denaturing gel electrophoresis and stained using ( D ) Coomassie blue or ( E ) SYBR Gold, respectively. The phosphorylated form of SF1 1–255 runs higher by SDS-PAGE. STD, size markers; nt; nucleotides. Conditions were identical to those of fluorescence assays with the exception that the RNase inhibitor <t>Superase-In</t> (Ambion, Life Technologies) was omitted here to fully rule out RNase contamination.
    Superase In, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "SF1 Phosphorylation Enhances Specific Binding to U2AF65 and Reduces Binding to 3′-Splice-Site RNA"

    Article Title: SF1 Phosphorylation Enhances Specific Binding to U2AF65 and Reduces Binding to 3′-Splice-Site RNA

    Journal: Biophysical Journal

    doi: 10.1016/j.bpj.2016.11.007

    SF1 structure and function in pre-mRNA splicing. ( A ) Schematic diagram of the early-stage SF1-U2AF 65 -U2AF 35 splicing-factor complex recognizing the 3′ splice site. ULMs are represented by “W.” White circles represent UHMK1-phosphorylation sites. BPS, branch point sequence; Py, polypyrimidine. ( B ) Domains of SF1, U2AF 65 , and U2AF 35 splicing factors. The boundaries of constructs used in this study are indicated by double-headed arrows. Zn, CCCH zinc knuckle; UHM, U2AF homology motif; ULM, U2AF ligand motif; RRM, RNA recognition motif, RS, arginine-serine repeat; KH-QUA2, K homology with quaking-2 motif; PA, protein A; circled P, UHMK1-phosphorylation sites. ( C ) Structure of the phosphorylated SF1 14–132 -U2AF 65 ). A line representing residues that are absent from the electron density connects the C-terminus of the ULM to the coiled-coil motifs. The tryptophan of the ULM (W22) and phosphorylated serines are shown as spheres and arginine residues that are mutated in this study are shown in stick representation. The inset shows a sequence alignment of the UHMK1-phosphorylated SPSP motif ( bold (between lines)). ( D and E ) Samples of ( D ) phospho-SF1 1–255 and ( E ) AdML L RNA under experimental conditions. Samples either before ( Pre- ) or after ( Post- ) incubating at 23°C for 2 h in the presence of a 10-fold excess of SF1 (U) or phospho-SF1 (P) were analyzed by denaturing gel electrophoresis and stained using ( D ) Coomassie blue or ( E ) SYBR Gold, respectively. The phosphorylated form of SF1 1–255 runs higher by SDS-PAGE. STD, size markers; nt; nucleotides. Conditions were identical to those of fluorescence assays with the exception that the RNase inhibitor Superase-In (Ambion, Life Technologies) was omitted here to fully rule out RNase contamination.
    Figure Legend Snippet: SF1 structure and function in pre-mRNA splicing. ( A ) Schematic diagram of the early-stage SF1-U2AF 65 -U2AF 35 splicing-factor complex recognizing the 3′ splice site. ULMs are represented by “W.” White circles represent UHMK1-phosphorylation sites. BPS, branch point sequence; Py, polypyrimidine. ( B ) Domains of SF1, U2AF 65 , and U2AF 35 splicing factors. The boundaries of constructs used in this study are indicated by double-headed arrows. Zn, CCCH zinc knuckle; UHM, U2AF homology motif; ULM, U2AF ligand motif; RRM, RNA recognition motif, RS, arginine-serine repeat; KH-QUA2, K homology with quaking-2 motif; PA, protein A; circled P, UHMK1-phosphorylation sites. ( C ) Structure of the phosphorylated SF1 14–132 -U2AF 65 ). A line representing residues that are absent from the electron density connects the C-terminus of the ULM to the coiled-coil motifs. The tryptophan of the ULM (W22) and phosphorylated serines are shown as spheres and arginine residues that are mutated in this study are shown in stick representation. The inset shows a sequence alignment of the UHMK1-phosphorylated SPSP motif ( bold (between lines)). ( D and E ) Samples of ( D ) phospho-SF1 1–255 and ( E ) AdML L RNA under experimental conditions. Samples either before ( Pre- ) or after ( Post- ) incubating at 23°C for 2 h in the presence of a 10-fold excess of SF1 (U) or phospho-SF1 (P) were analyzed by denaturing gel electrophoresis and stained using ( D ) Coomassie blue or ( E ) SYBR Gold, respectively. The phosphorylated form of SF1 1–255 runs higher by SDS-PAGE. STD, size markers; nt; nucleotides. Conditions were identical to those of fluorescence assays with the exception that the RNase inhibitor Superase-In (Ambion, Life Technologies) was omitted here to fully rule out RNase contamination.

    Techniques Used: Sequencing, Construct, Nucleic Acid Electrophoresis, Staining, SDS Page, Fluorescence

    Related Articles

    Centrifugation:

    Article Title: Ultra-high throughput single-cell RNA sequencing by combinatorial fluidic indexing
    Article Snippet: .. Preparation of permeabilized cell suspension A total of 5 million cells were washed with 10 ml of ice-cold 1x PBS (Gibco #14190-094) with centrifugation (300 rcf, 5 min, 4 °C) and fixed in 5 ml of ice-cold methanol (Fisher Scientific #M/4000/17) at -20 °C for 10 min. After two additional washes (centrifugation: 300 rcf, 5 min, 4 °C) with 5 ml of ice-cold PBS-BSA-SUPERase (1x PBS supplemented with 1% w/v BSA (Sigma #A8806-5) and 1% v/v SUPERase-In RNase Inhibitor (Thermo Fisher Scientific #AM2696)) permeabilized cells were resuspended in 200 µl of ice-cold PBS-BSA-SUPERase, and filtered through a cell strainer (40 µM or 70 µM depending on the cell size). .. We then used 5 µl of the sample for cell counting in duplicates on a CASY device (Schärfe System) and diluted to 5,000 cells per µl with ice-cold PBS-BSA-SUPERase.

    Flow Cytometry:

    Article Title: KSHV induces immunoglobulin rearrangements in mature B lymphocytes
    Article Snippet: .. Single cell RT-PCR for immunoglobulin light chains Single cells were harvested by flow sorting into 96-well PCR plates containing 4μl of RNA lysis buffer (0.5x PBS+10mM DTT+4U SUPERas-In (Thermo Cat #AM2694)). .. Plates were sealed and stored at -80°C. cDNA was synthesized directly in wells using Thermo High Capacity cDNA synthesis kit (Cat #4368814).

    Article Title: KSHV induces immunoglobulin rearrangements in mature B lymphocytes
    Article Snippet: .. Single cells were harvested by flow sorting into 96-well PCR plates containing 4μl of RNA lysis buffer (0.5x PBS+10mM DTT+4U SUPERas-In (Thermo Cat #AM2694)). .. Plates were sealed and stored at -80°C. cDNA was synthesized directly in wells using Thermo High Capacity cDNA synthesis kit (Cat #4368814).

    Mouse Assay:

    Article Title: Epigenetic regulation of brain region-specific microglia clearance activity
    Article Snippet: .. Mice were euthanized with CO2 and brain regions from Cx3cr1CreErt2/+(Litt) ;Eef1a1LSL.eGFPL10a/+ mice were dissected, cut into small pieces and homogenized by manual compression between 2 glass slides in Hank’s balanced salt solution (HBSS) (141175-095, Invitrogen Corporation, Carlsbad, CA) supplemented with 0.05% Collagenase D (1-088-874, Roche, Basel, CH), 25 mU/ml DNase I (Sigma), 0.5% Dispase II (165859, Roche), 10 μl/ml RNasin (N2515, Promega, Madison, WI) and Superasin (AM2694, Applied Biosystems, Waltham, MA). .. The digestion reaction was incubated at 37°C for 30 minutes with tituration through a 5mL pipette every 10 minutes and was stopped by addition of EDTA to a final concentration of 1mM.

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: KSHV induces immunoglobulin rearrangements in mature B lymphocytes
    Article Snippet: .. Single cell RT-PCR for immunoglobulin light chains Single cells were harvested by flow sorting into 96-well PCR plates containing 4μl of RNA lysis buffer (0.5x PBS+10mM DTT+4U SUPERas-In (Thermo Cat #AM2694)). .. Plates were sealed and stored at -80°C. cDNA was synthesized directly in wells using Thermo High Capacity cDNA synthesis kit (Cat #4368814).

    Staining:

    Article Title: Selective ribosome profiling as a tool to study the interaction of chaperones and targeting factors with nascent polypeptide chains and ribosomes
    Article Snippet: .. 1-Ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC, Thermo Scientific, 22980) Glycine (Roth, 3790) NaHCO3 (Roth, 6885) Superase•In RNase inhibitor (Ambion, AM2696) Micrococcal nuclease (Nuclease S7) (Roche, 10107921001 or ) Ethylene glycol tetraacetic acid (EGTA, AppliChem, A0878) Sucrose (Sigma, 16104) Strep-Tactin Sepharose 50% suspension (IBA, 2-1201-025) DNA, sodium salt from salmon testes (Sigma-Aldrich, D1626) Kanamycin (Applichem, A1493) Isopropyl β-D-1-thiogalactopyranoside (IPTG, Roth, CN08) Colloidal coomassie staining solution (Roth, Roti-Blue quick, 4829) Imidazole (Roth, 3899) CAUTION Imidazole is corrosive. ..

    Polymerase Chain Reaction:

    Article Title: KSHV induces immunoglobulin rearrangements in mature B lymphocytes
    Article Snippet: .. Single cell RT-PCR for immunoglobulin light chains Single cells were harvested by flow sorting into 96-well PCR plates containing 4μl of RNA lysis buffer (0.5x PBS+10mM DTT+4U SUPERas-In (Thermo Cat #AM2694)). .. Plates were sealed and stored at -80°C. cDNA was synthesized directly in wells using Thermo High Capacity cDNA synthesis kit (Cat #4368814).

    Article Title: KSHV induces immunoglobulin rearrangements in mature B lymphocytes
    Article Snippet: .. Single cells were harvested by flow sorting into 96-well PCR plates containing 4μl of RNA lysis buffer (0.5x PBS+10mM DTT+4U SUPERas-In (Thermo Cat #AM2694)). .. Plates were sealed and stored at -80°C. cDNA was synthesized directly in wells using Thermo High Capacity cDNA synthesis kit (Cat #4368814).

    Lysis:

    Article Title: Analysis of FGF20-regulated genes in organ of Corti progenitors by translating ribosome affinity purification
    Article Snippet: .. Pooled ventral otocysts were homogenized in lysis buffer (20 mM HEPES KOH, pH 7.4, 150 mM KCl, 10 mM MgCl2 , EDTA-free protease inhibitors [Roche, 04693159001], 0.5 mM DTT, 100 µg/ml cycloheximide, 10 µl/ml rRNasin [Promega N2515], 10 µl/ml Superasin [Applied Biosystems, AM2696]) using a pre-chilled Kontes homogenizer (Kontes, 885512-0020). .. The supernatant (S2) was mixed with 1/8 volume of 10% NP-40 and 300 mM DHPC (reconstituted in lysis buffer; Avanti Polar Lipids 850306P) and incubated for 10 min on ice.

    Article Title: KSHV induces immunoglobulin rearrangements in mature B lymphocytes
    Article Snippet: .. Single cell RT-PCR for immunoglobulin light chains Single cells were harvested by flow sorting into 96-well PCR plates containing 4μl of RNA lysis buffer (0.5x PBS+10mM DTT+4U SUPERas-In (Thermo Cat #AM2694)). .. Plates were sealed and stored at -80°C. cDNA was synthesized directly in wells using Thermo High Capacity cDNA synthesis kit (Cat #4368814).

    Article Title: KSHV induces immunoglobulin rearrangements in mature B lymphocytes
    Article Snippet: .. Single cells were harvested by flow sorting into 96-well PCR plates containing 4μl of RNA lysis buffer (0.5x PBS+10mM DTT+4U SUPERas-In (Thermo Cat #AM2694)). .. Plates were sealed and stored at -80°C. cDNA was synthesized directly in wells using Thermo High Capacity cDNA synthesis kit (Cat #4368814).

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    Thermo Fisher superase• in rnase inhibitor
    A variety of B lymphocyte lineages from human tonsil are susceptible to infection with BAC16 KSHV. Magnetically sorted total B lymphocytes from four tonsil specimens were infected with KSHV or mock-infected and analyzed by FCM at indicated timepoints for (A) GFP expression and (B) immunophenotypic markers for lineage. In both cases, cells were gated for singlet/viable/CD19+. Memory B cells were further defined as CD38low/IgD-/CD27+, naïve B cells were CD38low/IgD+/CD27-, natural effector (Nat Effector) cells were CD38low/IgD+/CD27+ and germinal center (GC) cells were CD38hi/IgD-. (C) In similar infection experiments with four tonsil specimens, total <t>RNA</t> was extracted at 2, 4 and 6 days post-infection and viral gene transcription was verified in two technical replicates by <t>RT-PCR.</t> Replicate RT negative cDNA reactions for KSHV infected samples at 6 days post-infection were included as a control for DNA contamination and mean NRT Cq values (n = 8) for each target were as follows: 39.44 for LANA, 40.52 for ORF59 and > 40 (not detectable) for K8.1. For a 40-cycle reaction, non-amplifying samples were set to Cq = 41 for the purposes of calculation. The lowest Cq value obtained in a mock infected sample was assigned as the limit of detection for each target, and data points that fall below this threshold are denoted with red shading. Yellow shading highlights values between 1.7 and 3.3 cycles lower than the limit of detection and corresponds to 5–10 fold increases in gene expression. Green shading highlights values more than 3.3 cycles lower than the limit of detection and corresponds to gene expression levels greater than 10-fold above the limit of detection. ANOVA analysis of raw Cq values revealed a statistically significant effect of KSHV infection for all target genes when comparing aggregate trends for mock vs KSHV samples over time: LANA p = 0.0006; K8.1 p = 0.02, ORF59 p
    Superase• In Rnase Inhibitor, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 292 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/superase• in rnase inhibitor/product/Thermo Fisher
    Average 99 stars, based on 292 article reviews
    Price from $9.99 to $1999.99
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    Thermo Fisher superase in rnase inhibitor
    Unique binding motifs in the N-terminus of SLBP interact with FEM1A, FEM1B, and FEM1C. (A) Diagram representing the domain structure of SLBP. The amino acid sequence and substrate receptor binding motifs representing the “degron hotspot” are shown. TAD, translational activation domain; NLS, nuclear localization sequence; RBD, RNA binding domain. (B) FEM1A, FEM1B, and FEM1C interact with amino acids 1–99 of SLBP. C-E Mapping the FEM1A, FEM1B and FEM1C binding regions in SLBP. HEK293T cells were transfected with either empty vector (EV) or FS-tagged SLBP constructs. MLN4924 was added to the cells for 4 hours before collection. Cell lysates were affinity precipitated with anti-STREP resin, and affinity precipitations were probed with the indicated antibodies. (F) The ligase-deficient SLBP(ABCdegron) mutant is unable to bind to CTIF. HEK293T cells were transfected with FLAG-tagged SLBP constructs. Cell lysates were supplemented with <t>SUPERase-In™</t> <t>RNase</t> Inhibitor and immunoprecipitated with anti-FLAG resin. The immunoprecipitations were probed with the indicated antibodies.
    Superase In Rnase Inhibitor, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 99 stars, based on 11 article reviews
    Price from $9.99 to $1999.99
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    A variety of B lymphocyte lineages from human tonsil are susceptible to infection with BAC16 KSHV. Magnetically sorted total B lymphocytes from four tonsil specimens were infected with KSHV or mock-infected and analyzed by FCM at indicated timepoints for (A) GFP expression and (B) immunophenotypic markers for lineage. In both cases, cells were gated for singlet/viable/CD19+. Memory B cells were further defined as CD38low/IgD-/CD27+, naïve B cells were CD38low/IgD+/CD27-, natural effector (Nat Effector) cells were CD38low/IgD+/CD27+ and germinal center (GC) cells were CD38hi/IgD-. (C) In similar infection experiments with four tonsil specimens, total RNA was extracted at 2, 4 and 6 days post-infection and viral gene transcription was verified in two technical replicates by RT-PCR. Replicate RT negative cDNA reactions for KSHV infected samples at 6 days post-infection were included as a control for DNA contamination and mean NRT Cq values (n = 8) for each target were as follows: 39.44 for LANA, 40.52 for ORF59 and > 40 (not detectable) for K8.1. For a 40-cycle reaction, non-amplifying samples were set to Cq = 41 for the purposes of calculation. The lowest Cq value obtained in a mock infected sample was assigned as the limit of detection for each target, and data points that fall below this threshold are denoted with red shading. Yellow shading highlights values between 1.7 and 3.3 cycles lower than the limit of detection and corresponds to 5–10 fold increases in gene expression. Green shading highlights values more than 3.3 cycles lower than the limit of detection and corresponds to gene expression levels greater than 10-fold above the limit of detection. ANOVA analysis of raw Cq values revealed a statistically significant effect of KSHV infection for all target genes when comparing aggregate trends for mock vs KSHV samples over time: LANA p = 0.0006; K8.1 p = 0.02, ORF59 p

    Journal: PLoS Pathogens

    Article Title: KSHV induces immunoglobulin rearrangements in mature B lymphocytes

    doi: 10.1371/journal.ppat.1006967

    Figure Lengend Snippet: A variety of B lymphocyte lineages from human tonsil are susceptible to infection with BAC16 KSHV. Magnetically sorted total B lymphocytes from four tonsil specimens were infected with KSHV or mock-infected and analyzed by FCM at indicated timepoints for (A) GFP expression and (B) immunophenotypic markers for lineage. In both cases, cells were gated for singlet/viable/CD19+. Memory B cells were further defined as CD38low/IgD-/CD27+, naïve B cells were CD38low/IgD+/CD27-, natural effector (Nat Effector) cells were CD38low/IgD+/CD27+ and germinal center (GC) cells were CD38hi/IgD-. (C) In similar infection experiments with four tonsil specimens, total RNA was extracted at 2, 4 and 6 days post-infection and viral gene transcription was verified in two technical replicates by RT-PCR. Replicate RT negative cDNA reactions for KSHV infected samples at 6 days post-infection were included as a control for DNA contamination and mean NRT Cq values (n = 8) for each target were as follows: 39.44 for LANA, 40.52 for ORF59 and > 40 (not detectable) for K8.1. For a 40-cycle reaction, non-amplifying samples were set to Cq = 41 for the purposes of calculation. The lowest Cq value obtained in a mock infected sample was assigned as the limit of detection for each target, and data points that fall below this threshold are denoted with red shading. Yellow shading highlights values between 1.7 and 3.3 cycles lower than the limit of detection and corresponds to 5–10 fold increases in gene expression. Green shading highlights values more than 3.3 cycles lower than the limit of detection and corresponds to gene expression levels greater than 10-fold above the limit of detection. ANOVA analysis of raw Cq values revealed a statistically significant effect of KSHV infection for all target genes when comparing aggregate trends for mock vs KSHV samples over time: LANA p = 0.0006; K8.1 p = 0.02, ORF59 p

    Article Snippet: Single cells were harvested by flow sorting into 96-well PCR plates containing 4μl of RNA lysis buffer (0.5x PBS+10mM DTT+4U SUPERas-In (Thermo Cat #AM2694)).

    Techniques: Infection, Expressing, Reverse Transcription Polymerase Chain Reaction

    A variety of B lymphocyte lineages from human tonsil are susceptible to infection with BAC16 KSHV. Magnetically sorted total B lymphocytes from four tonsil specimens were infected with KSHV or mock-infected and analyzed by FCM at indicated timepoints for (A) GFP expression and (B) immunophenotypic markers for lineage. In both cases, cells were gated for singlet/viable/CD19+. Memory B cells were further defined as CD38low/IgD-/CD27+, naïve B cells were CD38low/IgD+/CD27-, natural effector (Nat Effector) cells were CD38low/IgD+/CD27+ and germinal center (GC) cells were CD38hi/IgD-. (C) In similar infection experiments with four tonsil specimens, total RNA was extracted at 2, 4 and 6 days post-infection and viral gene transcription was verified in two technical replicates by RT-PCR. Replicate RT negative cDNA reactions for KSHV infected samples at 6 days post-infection were included as a control for DNA contamination and mean NRT Cq values (n = 8) for each target were as follows: 39.44 for LANA, 40.52 for ORF59 and > 40 (not detectable) for K8.1. For a 40-cycle reaction, non-amplifying samples were set to Cq = 41 for the purposes of calculation. The lowest Cq value obtained in a mock infected sample was assigned as the limit of detection for each target, and data points that fall below this threshold are denoted with red shading. Yellow shading highlights values between 1.7 and 3.3 cycles lower than the limit of detection and corresponds to 5–10 fold increases in gene expression. Green shading highlights values more than 3.3 cycles lower than the limit of detection and corresponds to gene expression levels greater than 10-fold above the limit of detection. ANOVA analysis of raw Cq values revealed a statistically significant effect of KSHV infection for all target genes when comparing aggregate trends for mock vs KSHV samples over time: LANA p = 0.0006; K8.1 p = 0.02, ORF59 p

    Journal: PLoS Pathogens

    Article Title: KSHV induces immunoglobulin rearrangements in mature B lymphocytes

    doi: 10.1371/journal.ppat.1006967

    Figure Lengend Snippet: A variety of B lymphocyte lineages from human tonsil are susceptible to infection with BAC16 KSHV. Magnetically sorted total B lymphocytes from four tonsil specimens were infected with KSHV or mock-infected and analyzed by FCM at indicated timepoints for (A) GFP expression and (B) immunophenotypic markers for lineage. In both cases, cells were gated for singlet/viable/CD19+. Memory B cells were further defined as CD38low/IgD-/CD27+, naïve B cells were CD38low/IgD+/CD27-, natural effector (Nat Effector) cells were CD38low/IgD+/CD27+ and germinal center (GC) cells were CD38hi/IgD-. (C) In similar infection experiments with four tonsil specimens, total RNA was extracted at 2, 4 and 6 days post-infection and viral gene transcription was verified in two technical replicates by RT-PCR. Replicate RT negative cDNA reactions for KSHV infected samples at 6 days post-infection were included as a control for DNA contamination and mean NRT Cq values (n = 8) for each target were as follows: 39.44 for LANA, 40.52 for ORF59 and > 40 (not detectable) for K8.1. For a 40-cycle reaction, non-amplifying samples were set to Cq = 41 for the purposes of calculation. The lowest Cq value obtained in a mock infected sample was assigned as the limit of detection for each target, and data points that fall below this threshold are denoted with red shading. Yellow shading highlights values between 1.7 and 3.3 cycles lower than the limit of detection and corresponds to 5–10 fold increases in gene expression. Green shading highlights values more than 3.3 cycles lower than the limit of detection and corresponds to gene expression levels greater than 10-fold above the limit of detection. ANOVA analysis of raw Cq values revealed a statistically significant effect of KSHV infection for all target genes when comparing aggregate trends for mock vs KSHV samples over time: LANA p = 0.0006; K8.1 p = 0.02, ORF59 p

    Article Snippet: Single cell RT-PCR for immunoglobulin light chains Single cells were harvested by flow sorting into 96-well PCR plates containing 4μl of RNA lysis buffer (0.5x PBS+10mM DTT+4U SUPERas-In (Thermo Cat #AM2694)).

    Techniques: Infection, Expressing, Reverse Transcription Polymerase Chain Reaction

    scifi-RNA-seq alignment metrics for whole cells, fresh nuclei, and fixed nuclei a) 15,300 pre-indexed nuclei from a mixture of human (Jurkat) and mouse (3T3) cells were processed in a single microfluidic channel and demultiplexed based on the microfluidic round2 barcode only (left plot), or based on the combination of round1 and round2 barcodes (right plot). At the standard loading concentration of the Chromium device (15,300 nuclei per channel), the microfluidic (round2) index provides sufficient complexity to resolve single cells, although the combination of round1 and round2 barcodes still results in a reduction of background noise. b) Coverage along human and mouse transcripts from 200 bp upstream of the transcription start site (TSS) to 200 bp downstream of the transcription end site (TES), shown for whole cells permeabilized by methanol, freshly isolated nuclei, and nuclei fixed with 1% or 4% formaldehyde that were cryopreserved, re-hydrated, and permeabilized. Freshly isolated nuclei show the strongest 3’ enrichment. c) Boxplots summarizing sequence alignment metrics across the different types of input material: Total reads sequenced, percent uniquely mapped reads, percent multi-mappers, percent alignments to exons plus introns, percent alignments to exons, and percent spliced reads. Freshly isolated nuclei showed the best performance for these alignment metrics.

    Journal: bioRxiv

    Article Title: Ultra-high throughput single-cell RNA sequencing by combinatorial fluidic indexing

    doi: 10.1101/2019.12.17.879304

    Figure Lengend Snippet: scifi-RNA-seq alignment metrics for whole cells, fresh nuclei, and fixed nuclei a) 15,300 pre-indexed nuclei from a mixture of human (Jurkat) and mouse (3T3) cells were processed in a single microfluidic channel and demultiplexed based on the microfluidic round2 barcode only (left plot), or based on the combination of round1 and round2 barcodes (right plot). At the standard loading concentration of the Chromium device (15,300 nuclei per channel), the microfluidic (round2) index provides sufficient complexity to resolve single cells, although the combination of round1 and round2 barcodes still results in a reduction of background noise. b) Coverage along human and mouse transcripts from 200 bp upstream of the transcription start site (TSS) to 200 bp downstream of the transcription end site (TES), shown for whole cells permeabilized by methanol, freshly isolated nuclei, and nuclei fixed with 1% or 4% formaldehyde that were cryopreserved, re-hydrated, and permeabilized. Freshly isolated nuclei show the strongest 3’ enrichment. c) Boxplots summarizing sequence alignment metrics across the different types of input material: Total reads sequenced, percent uniquely mapped reads, percent multi-mappers, percent alignments to exons plus introns, percent alignments to exons, and percent spliced reads. Freshly isolated nuclei showed the best performance for these alignment metrics.

    Article Snippet: Preparation of permeabilized cell suspension A total of 5 million cells were washed with 10 ml of ice-cold 1x PBS (Gibco #14190-094) with centrifugation (300 rcf, 5 min, 4 °C) and fixed in 5 ml of ice-cold methanol (Fisher Scientific #M/4000/17) at -20 °C for 10 min. After two additional washes (centrifugation: 300 rcf, 5 min, 4 °C) with 5 ml of ice-cold PBS-BSA-SUPERase (1x PBS supplemented with 1% w/v BSA (Sigma #A8806-5) and 1% v/v SUPERase-In RNase Inhibitor (Thermo Fisher Scientific #AM2696)) permeabilized cells were resuspended in 200 µl of ice-cold PBS-BSA-SUPERase, and filtered through a cell strainer (40 µM or 70 µM depending on the cell size).

    Techniques: RNA Sequencing Assay, Concentration Assay, Isolation, Sequencing

    scifi-RNA-seq combines pre-indexing of whole transcriptomes with droplet-based scRNA-seq a) Standard droplet-based scRNA-seq is highly inefficient in its reagent usage. To avoid cell doublets, the single cell suspension is loaded into the microfluidic device at a very low concentration (limiting dilution), leaving most droplets without a cell. Even though the vast majority of droplets contain both a barcoded microbead and reverse transcription reagents, and are thus fully functional, they are not used to generate transcriptome data. Even in droplets that receive one cell, the reagents could in principle support barcoding of additional cells, which adds to the inefficiency. b) scifi-RNA-seq uses pre-indexing and droplet overloading to boost the throughput of droplet-based scRNA-seq. Prior to microfluidic indexing, cells or nuclei are permeabilized and their whole transcriptomes are pre-indexed with a well-specific (round1) barcode by reverse transcription on a multiwell plate. Intact cells or nuclei containing differentially barcoded cDNA are randomly mixed and encapsulated in droplets at high concentration using a standard microfluidic droplet generator, such that most droplets contain multiple cells or nuclei (droplet overloading). Inside the droplets, a droplet-specific microfluidic (round2) barcode is added to the pre-indexed cDNA molecules by thermoligation. The combination of round1 and round2 barcodes uniquely identifies single cells. c) Detailed method design for scifi-RNA-seq. d) By omitting the lysis reagents, intact nuclei can be imaged inside emulsion droplets, confirming the feasibility of droplet overloading using a microfluidic droplet generator (10x Genomics Chromium). Representative droplets containing between one and ten nuclei are shown. e) Droplet overloading boosts the percentage of droplets filled with nuclei from 16.4% (maximum loading concentration according to the standard Chromium protocol) to 95.5% (100-fold overloading using 1.53 million nuclei per channel). f) Droplet overloading causes the average number of nuclei per droplet to increase in a controlled fashion while maintaining the desired Poisson-like loading distribution. g) Expected collision rate as a function of the cell/nuclei loading concentration per channel for defined sets of round1 barcodes. The cell/nuclei fill rate was modelled as a zero-inflated Poisson distribution. h) Due to the high number of microfluidic round2 barcodes, two-round scifi-RNA-seq exceeds the barcoding capability of existing three-round combinatorial indexing protocols, while providing a more efficient and greatly simplified workflow.

    Journal: bioRxiv

    Article Title: Ultra-high throughput single-cell RNA sequencing by combinatorial fluidic indexing

    doi: 10.1101/2019.12.17.879304

    Figure Lengend Snippet: scifi-RNA-seq combines pre-indexing of whole transcriptomes with droplet-based scRNA-seq a) Standard droplet-based scRNA-seq is highly inefficient in its reagent usage. To avoid cell doublets, the single cell suspension is loaded into the microfluidic device at a very low concentration (limiting dilution), leaving most droplets without a cell. Even though the vast majority of droplets contain both a barcoded microbead and reverse transcription reagents, and are thus fully functional, they are not used to generate transcriptome data. Even in droplets that receive one cell, the reagents could in principle support barcoding of additional cells, which adds to the inefficiency. b) scifi-RNA-seq uses pre-indexing and droplet overloading to boost the throughput of droplet-based scRNA-seq. Prior to microfluidic indexing, cells or nuclei are permeabilized and their whole transcriptomes are pre-indexed with a well-specific (round1) barcode by reverse transcription on a multiwell plate. Intact cells or nuclei containing differentially barcoded cDNA are randomly mixed and encapsulated in droplets at high concentration using a standard microfluidic droplet generator, such that most droplets contain multiple cells or nuclei (droplet overloading). Inside the droplets, a droplet-specific microfluidic (round2) barcode is added to the pre-indexed cDNA molecules by thermoligation. The combination of round1 and round2 barcodes uniquely identifies single cells. c) Detailed method design for scifi-RNA-seq. d) By omitting the lysis reagents, intact nuclei can be imaged inside emulsion droplets, confirming the feasibility of droplet overloading using a microfluidic droplet generator (10x Genomics Chromium). Representative droplets containing between one and ten nuclei are shown. e) Droplet overloading boosts the percentage of droplets filled with nuclei from 16.4% (maximum loading concentration according to the standard Chromium protocol) to 95.5% (100-fold overloading using 1.53 million nuclei per channel). f) Droplet overloading causes the average number of nuclei per droplet to increase in a controlled fashion while maintaining the desired Poisson-like loading distribution. g) Expected collision rate as a function of the cell/nuclei loading concentration per channel for defined sets of round1 barcodes. The cell/nuclei fill rate was modelled as a zero-inflated Poisson distribution. h) Due to the high number of microfluidic round2 barcodes, two-round scifi-RNA-seq exceeds the barcoding capability of existing three-round combinatorial indexing protocols, while providing a more efficient and greatly simplified workflow.

    Article Snippet: Preparation of permeabilized cell suspension A total of 5 million cells were washed with 10 ml of ice-cold 1x PBS (Gibco #14190-094) with centrifugation (300 rcf, 5 min, 4 °C) and fixed in 5 ml of ice-cold methanol (Fisher Scientific #M/4000/17) at -20 °C for 10 min. After two additional washes (centrifugation: 300 rcf, 5 min, 4 °C) with 5 ml of ice-cold PBS-BSA-SUPERase (1x PBS supplemented with 1% w/v BSA (Sigma #A8806-5) and 1% v/v SUPERase-In RNase Inhibitor (Thermo Fisher Scientific #AM2696)) permeabilized cells were resuspended in 200 µl of ice-cold PBS-BSA-SUPERase, and filtered through a cell strainer (40 µM or 70 µM depending on the cell size).

    Techniques: RNA Sequencing Assay, Concentration Assay, Functional Assay, Lysis

    scifi-RNA-seq yields high-quality data for whole cells, fresh nuclei, and fixed nuclei We prepared a mixture of human (Jurkat) and mouse (3T3) cells, and performed scifi-RNA-seq on whole cells permeabilized by methanol, freshly isolated nuclei, and nuclei fixed with 1% or 4% formaldehyde that were cryopreserved, re-hydrated, and permeabilized. During reverse transcription on a 96-well plate, each sample was assigned a specific set of round1 barcodes. Afterward, all wells were pooled, and 15,300 cells/nuclei were loaded into a single channel of the Chromium device. We provide the following performance plots: (i) ranked barcodes plotted against reads, unique molecular identifiers (UMIs), or detected genes, distinguishing single-cell transcrip-tomes from background noise; (ii) reads plotted against UMIs; (iii) reads plotted against the number of detected genes; (iv) reads plotted against the fraction of unique reads; (v) species mixing plot showing the number of UMIs per cell aligning to the mouse genome (x-axis) versus the human genome (y-axis). To facilitate comparisons between different types of input material, the axes of the performance plots use the same scale across conditions.

    Journal: bioRxiv

    Article Title: Ultra-high throughput single-cell RNA sequencing by combinatorial fluidic indexing

    doi: 10.1101/2019.12.17.879304

    Figure Lengend Snippet: scifi-RNA-seq yields high-quality data for whole cells, fresh nuclei, and fixed nuclei We prepared a mixture of human (Jurkat) and mouse (3T3) cells, and performed scifi-RNA-seq on whole cells permeabilized by methanol, freshly isolated nuclei, and nuclei fixed with 1% or 4% formaldehyde that were cryopreserved, re-hydrated, and permeabilized. During reverse transcription on a 96-well plate, each sample was assigned a specific set of round1 barcodes. Afterward, all wells were pooled, and 15,300 cells/nuclei were loaded into a single channel of the Chromium device. We provide the following performance plots: (i) ranked barcodes plotted against reads, unique molecular identifiers (UMIs), or detected genes, distinguishing single-cell transcrip-tomes from background noise; (ii) reads plotted against UMIs; (iii) reads plotted against the number of detected genes; (iv) reads plotted against the fraction of unique reads; (v) species mixing plot showing the number of UMIs per cell aligning to the mouse genome (x-axis) versus the human genome (y-axis). To facilitate comparisons between different types of input material, the axes of the performance plots use the same scale across conditions.

    Article Snippet: Preparation of permeabilized cell suspension A total of 5 million cells were washed with 10 ml of ice-cold 1x PBS (Gibco #14190-094) with centrifugation (300 rcf, 5 min, 4 °C) and fixed in 5 ml of ice-cold methanol (Fisher Scientific #M/4000/17) at -20 °C for 10 min. After two additional washes (centrifugation: 300 rcf, 5 min, 4 °C) with 5 ml of ice-cold PBS-BSA-SUPERase (1x PBS supplemented with 1% w/v BSA (Sigma #A8806-5) and 1% v/v SUPERase-In RNase Inhibitor (Thermo Fisher Scientific #AM2696)) permeabilized cells were resuspended in 200 µl of ice-cold PBS-BSA-SUPERase, and filtered through a cell strainer (40 µM or 70 µM depending on the cell size).

    Techniques: RNA Sequencing Assay, Isolation

    Unique binding motifs in the N-terminus of SLBP interact with FEM1A, FEM1B, and FEM1C. (A) Diagram representing the domain structure of SLBP. The amino acid sequence and substrate receptor binding motifs representing the “degron hotspot” are shown. TAD, translational activation domain; NLS, nuclear localization sequence; RBD, RNA binding domain. (B) FEM1A, FEM1B, and FEM1C interact with amino acids 1–99 of SLBP. C-E Mapping the FEM1A, FEM1B and FEM1C binding regions in SLBP. HEK293T cells were transfected with either empty vector (EV) or FS-tagged SLBP constructs. MLN4924 was added to the cells for 4 hours before collection. Cell lysates were affinity precipitated with anti-STREP resin, and affinity precipitations were probed with the indicated antibodies. (F) The ligase-deficient SLBP(ABCdegron) mutant is unable to bind to CTIF. HEK293T cells were transfected with FLAG-tagged SLBP constructs. Cell lysates were supplemented with SUPERase-In™ RNase Inhibitor and immunoprecipitated with anti-FLAG resin. The immunoprecipitations were probed with the indicated antibodies.

    Journal: Cell Cycle

    Article Title: FEM1 proteins are ancient regulators of SLBP degradation

    doi: 10.1080/15384101.2017.1284715

    Figure Lengend Snippet: Unique binding motifs in the N-terminus of SLBP interact with FEM1A, FEM1B, and FEM1C. (A) Diagram representing the domain structure of SLBP. The amino acid sequence and substrate receptor binding motifs representing the “degron hotspot” are shown. TAD, translational activation domain; NLS, nuclear localization sequence; RBD, RNA binding domain. (B) FEM1A, FEM1B, and FEM1C interact with amino acids 1–99 of SLBP. C-E Mapping the FEM1A, FEM1B and FEM1C binding regions in SLBP. HEK293T cells were transfected with either empty vector (EV) or FS-tagged SLBP constructs. MLN4924 was added to the cells for 4 hours before collection. Cell lysates were affinity precipitated with anti-STREP resin, and affinity precipitations were probed with the indicated antibodies. (F) The ligase-deficient SLBP(ABCdegron) mutant is unable to bind to CTIF. HEK293T cells were transfected with FLAG-tagged SLBP constructs. Cell lysates were supplemented with SUPERase-In™ RNase Inhibitor and immunoprecipitated with anti-FLAG resin. The immunoprecipitations were probed with the indicated antibodies.

    Article Snippet: SUPERase-In™ RNase Inhibitor (Thermo Fisher Scientific) was used at 1U/μL where indicated.

    Techniques: Binding Assay, Sequencing, Activation Assay, RNA Binding Assay, Transfection, Plasmid Preparation, Construct, Mutagenesis, Immunoprecipitation