dgcr8 knockout mefs  (Novus Biologicals)

Journal: Cell stem cell

Article Title: A CLK3-HMGA2 Alternative Splicing Axis Impacts Human Hematopoietic Stem Cell Molecular Identity throughout Development.

doi: 10.1016/j.stem.2018.03.012

Figure Lengend Snippet: Figure 4. Post-transcriptional Regulation of HMGA2 Isoforms (A) Left: Normalized luciferase (Renilla) activity in Dgcr8-KO MEF of constructs carrying 30 UTR sequences of HMGA2-L (Rluc-30UTRwt_HMGA2-L) or a mutant derivative depleted for miRNA sites (Rluc-30UTRmt_HMGA2-L). Right: Normalized luciferase (Renilla) activity in Dgcr8-KO MEF of constructs carrying 30 UTR sequences of HMGA2-L (Rluc-30UTRwt_HMGA2-L) or HMGA2-S (Rluc-30UTRwt_HMGA2-S). Normalized luciferase activities were reported with respect to Rluc-30UTRmt_HMGA2-L (left) or Rluc-30UTRwt_HMGA2-S (right), set to 100%. Mean ± SEM values are shown. Unpaired t test was used; *p < 0.05, **p < 0.01, ***p < 0.005, borderline (०= 0.055). (B) Quantification of miRNAs of interest in PC-3 and HPC-5F cells measured by qRT-PCR. U6 small nuclear RNA (snRNA) was used as control. (C) Relative quantification of HMGA2 isoforms in PC-3 and HPC-5F cells transduced with lentiviral constructs carrying the HMGA2 ORFs equipped with their corresponding 30 UTRs (HMGA2-L+30UTRwt and HMGA2-S+30UTRwt) or a derivative HMGA2-L isoform mutated at its miRNA sites (HMGA2-L+30UTRmt). Infected cells were treated with actinomycin D (Act-D) and harvested at the indicated time points. Expression values were normalized to HPRT1 control and then reported with respect to HMGA2-S+30UTRwt, set to 1. Mean ± SEM values are shown. ANOVA was used; *p < 0.05, **p < 0.01, ***p < 0.005.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies CD34 PE-Cy7 BD Biosciences Cat. 348791; CLONE 8G12 CD38 PE-Cy5 BD Biosciences Cat. 555461; CLONE HIT2 CD90 PE BD Biosciences Cat. 555596; CLONE 5E10 CD45RA FITC BioLegend Cat. 304105; CLONE HI100 DAPI solution BD Biosciences Cat. 564907 CD45RA V450 BD Biosciences Cat. 560362; CLONE HI100 CD133/1 APC Miltenyi Biotec Cat. 130-090-826; CLONE AC133 CD19 PE BD Biosciences Cat. 349209; CLONE 4G7 CD45 PE-Cy5 Coulter CLONE Immu19.2 CD33 APC BD Biosciences Cat. 340474; CLONE P67.6 CD45 APC-Cy7 BD Biosciences Cat. 561863; CLONE 2D1 V5 MBL Cat. M167-3 H3K4me2 Diagenode Cat. 035-050 Biological Samples Cord Blood CD34+ Cells Lonza Cat. 2C-101 Cord Blood CD34+ Cells AllCells Cat. CB008F Fetal Liver CD34+ Cells AllCells Cat. FL-CD34-002F Bone Marrow CD34+ Cells AllCells Cat. ABM017F Bone Marrow CD34+ Cells Lonza Cat. 2M-101C Chemicals, Peptides, and Recombinant Proteins Actinomycin D Sigma-Aldrich Cat. A1410 Doxycycline Hyclate Sigma-Aldrich Cat. D9891 Protamine Sulfate Sigma-Aldrich Cat. P4020 Recombinant Human SCF Peprotech Cat. 300-07 Recombinant Human FLT3L Peprotech Cat. 300-19 Recombinant Human TPO Peprotech Cat. 300-18 Recombinant Human IL6 Peprotech Cat. 200-06 Retronectin Clontech Cat. T100A DOTAP Liposomal Transfection Reagent Sigma-Aldrich Cat. 11202375001 DharmaFECT Duo Transfection Reagent Dharmacon Cat. T-2010-03 Critical Commercial Assays Dual-Luciferase Reporter Assay System Promega Cat. E1910 TruSeq RNA Library Prep Kit v2 Illumina Cat. RS-122-2001 SMART-Seq v4 Ultra Low Input RNA Kit Clontech Cat. 634888 Nextera XT DNA Library Preparation Kit Illumina Cat. FC-131-1024 nCounter Human v2 miRNA Expression Assay NanoString Technologies Cat. GXA-MIR2-24 ChiP-Seq Assay Broad Institute Epigenomics Program N/A Experimental Models: Cell Lines PC-3 ATCC Cat. CRL-1435 K562 ATCC Cat. CCL-243 HPC and HPC-5F Doulatov et al., 2013 N/A DGCR8 knockout MEF Novus Biologicals Cat. NBP2-25171 (Continued on next page) Cell Stem Cell 22, 575–588.e1–e7, April 5, 2018 e1

Techniques: Luciferase, Activity Assay, Construct, Mutagenesis, Quantitative RT-PCR, Control, Transduction, Infection, Expressing

Journal: Cell stem cell

Article Title: A CLK3-HMGA2 alternative splicing axis impacts human hematopoietic stem cell molecular identity throughout development

doi: 10.1016/j.stem.2018.03.012

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: DGCR8 knockout MEF , Novus Biologicals , Cat. NBP2-25171.

Techniques: Recombinant, Transfection, Reporter Assay, DNA Library Preparation, Expressing, Knock-Out, Negative Control, Software

Journal: Cell stem cell

Article Title: A CLK3-HMGA2 alternative splicing axis impacts human hematopoietic stem cell molecular identity throughout development

doi: 10.1016/j.stem.2018.03.012

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: DGCR8 knockout MEF , Novus Biologicals , Cat. NBP2-25171.

Techniques: Recombinant, Transfection, Reporter Assay, DNA Library Preparation, Expressing, Knock-Out, Negative Control, Software

Journal: Nucleic Acids Research

Article Title: Biogenesis of mammalian microRNAs by a non-canonical processing pathway

doi: 10.1093/nar/gks026

Figure Lengend Snippet: Simtron biogenesis involves Drosha but not DGCR8. Knockdown of DGCR8 in HeLa cells using siRNA was quantitated by ( A ) RT–PCR analysis of DGCR8 mRNA and ( B ) western blot analysis of DGCR8 protein expression. The percentage of knockdown of DGCR8 was quantitated for DGCR8 mRNA using the equation 100 − [(( DGCR8 knockdown / GAPDH )/( DGCR8 control / GAPDH )) × 100], n = 5 and for DGCR8 protein using the equation 100 − [(( DGCR8 knockdown /β-actin )/( DGCR8 control / β-actin )) × 100]. (C) Changes in endogenous miRNA levels following DGCR8 knockdown were analysed by stemloop RT–PCR analysis. miR-16 is a canonical miRNA control and sno65 is a loading control. Graph shows quantitation of miRNA abundance using the equation: (miRNA experimental condition /sno65)/(miRNA control /sno65). n = 4 for all miRNAs except for miR-16, n = 5; asterisk indicates P ≤ 0.05 (Wilcoxon matched pairs signed-rank test). M indicates a synthetic size marker and filled circle indicates a non-specific primer dimer. ( D ) RT–PCR analysis of Drosha mRNA following expression of TN-Drosha in HEK-293T cells. ( E ) The effect of TN-Drosha expression on endogenous miRNA abundance was analysed by stemloop RT–PCR. Graph shows quantitation of miRNA abundance using the same equation as in C, n = 6; asterick indicates P ≤ 0.05 (Student's t -test). ( F ) Stemloop RT–PCR analysis of minigene-derived miR-877, 1226, 1225, 1228 and endogenous miR-16 isolated from HEK-293T cells transiently transfected with TN-Drosha. sno65 was used as a control. TN-Drosha mRNA expression in HEK-293T cells was analysed by radiolabelled RT–PCR. GAPDH was used as a control. ( G ) Quantitation of miRNA abundance relative to sno65 using the equation: miRNA/sno65. n = 3 for miR-877, 1226 and 1225, n = 5 for miR-1228 and n = 14 for miR-16; * P ≤ 0.05, *** P ≤ 0.0001 (). Data sets were analysed using the Student's t -test with the exception of miR-16, which was analysed using the Wilcoxon matched pairs signed-rank test. In all panels, bars represent the average ± SEM. The horizontal dotted line indicates normalized control levels.

Article Snippet: Hannon) ( ) and DGCR8 knockout (DGCR8 −/− ) cells (Novus Biologicals) were grown on a gelatin layer in Knockout Dulbecco's modified Eagle's medium (Gibco) supplemented with 15% ES cell FBS (Gibco), 1% non-essential amino acids, 1% l -glutamine, 1% penicillin/streptomycin/Amphotericin B, 0.1% ESGRO-LIF and 0.008% beta-mercaptoethanol.

Techniques: Knockdown, Reverse Transcription Polymerase Chain Reaction, Western Blot, Expressing, Control, Quantitation Assay, Marker, Derivative Assay, Isolation, Transfection

Journal: Nucleic Acids Research

Article Title: Biogenesis of mammalian microRNAs by a non-canonical processing pathway

doi: 10.1093/nar/gks026

Figure Lengend Snippet: Simtron biogenesis does not require DGCR8, Dicer, Ago2 or XPO5. ( A ) RT–PCR analysis of minigene-derived host gene mRNA and stemloop RT–PCR analysis of minigene-derived miRNA and endogenous miR-16 in Dicer and DGCR8 knockout mouse embryonic stem cells transfected with the wt or splicing-deficient minigene (Δss) or empty vector control (−). sno65 was used as a loading control. Graphs show quantitation of miRNA using the equation: (miRNA experimental condition /sno65)/(miRNA control /sno65). Bars represent the average ± SEM, n = 3. The horizontal dotted lines indicate normalized control levels. ( B ) Stemloop RT–PCR analysis of miR-1225 and miR-1228 immunoprecipitated from HEK-293T cell lysates that were transiently transfected with wt or Δss minigenes, or miR-877 from wt minigene along with pFLAG-Dicer (Dicer) or without (−) and immunoprecipitated with an antibody against the FLAG epitope. Input refers to cell lysates before FLAG immunoprecipitation; Un is the unbound fraction and IP is the immunoprecipitated fraction. Un is 1/20 IP and Input is 1/5 IP. The graph represents the percent of the mature miRNA found in the IP fraction versus the amount that remained in the Un fraction using the equation: (IP/(IP + (Un × 20)) × 100). ( C ) Stemloop RT–PCR analysis of minigene-derived miR-1225, miR-1228 and endogenous miR-16 from Ago2 knockout mouse embryonic fibroblasts. sno65 was used as a loading control. Cells were transiently transfected with wt or Δss minigenes or empty vector control (−). Graph shows quantitation of miRNA abundance using the same equation as in A. Bars represent the average ± SEM, n = 3 and * P ≤ 0.05 or ** P ≤ 0.01, Student's t -test. The horizontal dotted lines indicate normalized control levels. ( D ) Stemloop RT–PCR and RT–PCR analysis of miR-877 (left panel), miR-1225 (middle panel) and miR-1228 (right panel) minigene-expression in HeLa cells following siRNA-directed knockdown of XPO5 . sno65 is a loading control for miRNA using stemloop RT–PCR and GAPDH is a loading control for RT–PCR of XPO5 mRNA.

Article Snippet: Hannon) ( ) and DGCR8 knockout (DGCR8 −/− ) cells (Novus Biologicals) were grown on a gelatin layer in Knockout Dulbecco's modified Eagle's medium (Gibco) supplemented with 15% ES cell FBS (Gibco), 1% non-essential amino acids, 1% l -glutamine, 1% penicillin/streptomycin/Amphotericin B, 0.1% ESGRO-LIF and 0.008% beta-mercaptoethanol.

Techniques: Reverse Transcription Polymerase Chain Reaction, Derivative Assay, Knock-Out, Transfection, Plasmid Preparation, Control, Quantitation Assay, Immunoprecipitation, FLAG-tag, Expressing, Knockdown

Journal: Nucleic Acids Research

Article Title: Biogenesis of mammalian microRNAs by a non-canonical processing pathway

doi: 10.1093/nar/gks026

Figure Lengend Snippet: Immunoprecipitation and in vitro processing of simtrons with Drosha. ( A ) Pre-miR-1225 co-immunoprecipitates with Drosha. Pre-miR-1225 derived from wt and Δss minigenes and pre-miR-877 from wt minigene were transiently transfected into HEK-293T cells with pFLAG-Drosha (Drosha) or without (−), and immunoprecipitated with an antibody against the FLAG epitope. Isolated pre-miRNAs were analysed by radiolabelled stemloop RT–PCR and products were separated by 12% native PAGE. Input (In) refers to cell lysates before FLAG immunoprecipitation; Un is the unbound fraction and IP is the immunoprecipitated fraction. Un is 1/20 IP and Input is 1/5 IP. The graph represents the percent of the pre-miRNA found in the IP fraction versus the amount that remained in the Un fraction using the equation: (IP/(IP + (Un × 20)) × 100). ( B ) Drosha-dependent in vitro simtron processing. Radiolabelled RNA transcribed from a PKD1 wt or Δss, ABCF1 wt or pri-miR-16-1 DNA template was incubated with the FLAG-immunoprecipitates from HEK-293T cells, or with HEK-293T WCEs from cells that were not transfected. FLAG-immunoprecipitates were derived from cells transfected with mock transfection (−), pFLAG-GFP (GFP), pFLAG-Drosha (Drosha), pFLAG-Drosha and pFLAG-DGCR8 (Drosha + DGCR8), pFLAG-TN-Drosha (TN Drosha), or FLAG-M2-beads that were incubated with lysis buffer but no cell lysate (−lysate). Template RNA was included as a control (RNA). Reaction products were separated by 8% denaturing PAGE. The sizes of pre-miRNAs are indicated. Asterisk indicates uncharacterized miR-16 cleavage fragments .

Article Snippet: Hannon) ( ) and DGCR8 knockout (DGCR8 −/− ) cells (Novus Biologicals) were grown on a gelatin layer in Knockout Dulbecco's modified Eagle's medium (Gibco) supplemented with 15% ES cell FBS (Gibco), 1% non-essential amino acids, 1% l -glutamine, 1% penicillin/streptomycin/Amphotericin B, 0.1% ESGRO-LIF and 0.008% beta-mercaptoethanol.

Techniques: Immunoprecipitation, In Vitro, Derivative Assay, Transfection, FLAG-tag, Isolation, Reverse Transcription Polymerase Chain Reaction, Clear Native PAGE, Incubation, Lysis, Control

Journal: Nucleic Acids Research

Article Title: Biogenesis of mammalian microRNAs by a non-canonical processing pathway

doi: 10.1093/nar/gks026

Figure Lengend Snippet: Simtron processing is context independent. ( A ) Diagram comparing intronic and intergenic pre-miRNA expression. ( B ) Control, Dicer (Dicer −/− ) or DGCR8 (DGCR8 −/− ) knockout mouse embryonic stem cells were transiently transfected with the intergenic wt minigene, or intergenic splicing-deficient minigene (Δss). Minigene-derived miRNAs and endogenous miR-16 were analysed by stemloop RT–PCR. Left panel: simtron miR-1225. Right panel: mirtron miR-877. sno65 was analysed as a loading control. ( C ) Graph shows quantitation of miR-1225 abundance using the equation: (miRNA Dicer−/− or DGCR8−/− /sno65)/(miRNA control /sno65). Bars represent the average values ±SEM, n = 4 for Dicer −/− and n = 3 for DGCR8 −/− . The horizontal dotted line indicates normalized control cell levels.

Article Snippet: Hannon) ( ) and DGCR8 knockout (DGCR8 −/− ) cells (Novus Biologicals) were grown on a gelatin layer in Knockout Dulbecco's modified Eagle's medium (Gibco) supplemented with 15% ES cell FBS (Gibco), 1% non-essential amino acids, 1% l -glutamine, 1% penicillin/streptomycin/Amphotericin B, 0.1% ESGRO-LIF and 0.008% beta-mercaptoethanol.

Techniques: Expressing, Control, Knock-Out, Transfection, Derivative Assay, Reverse Transcription Polymerase Chain Reaction, Quantitation Assay

Journal: Nucleic Acids Research

Article Title: Biogenesis of mammalian microRNAs by a non-canonical processing pathway

doi: 10.1093/nar/gks026

Figure Lengend Snippet: Proposed model of simtron biogenesis compared to other miRNA processing pathways. The pathways shown begin with the primary transcript and end with the mature product. Left: simtron pathway, Middle: mirtron pathway, Right: canonical miRNA pathway. Exons are depicted as boxes and introns and miRNAs as lines. Each protein or protein complex is labelled. Proteins labelled with question marks are proposed but not known. Simtrons (such as miR-1225 and miR-1228) processing from the intron involves Drosha and possibly an unknown binding partner. Simtrons are further processed by unknown factors and enter the RISC complex with any of the four human Argonaute proteins. Mirtrons (such as miR-877 and miR-1226) are excised from the host gene by the spliceosome, are debranched, exported from the nucleus by exportin5 (XPO5), cleaved by Dicer and enter the RISC complex. Canonical miRNAs (such as miR-16) are processed by Drosha and DGCR8, exported from the nucleus by XPO5, cleaved by Dicer and enter the RISC complex. All three pathways result in functional miRNAs.

Article Snippet: Hannon) ( ) and DGCR8 knockout (DGCR8 −/− ) cells (Novus Biologicals) were grown on a gelatin layer in Knockout Dulbecco's modified Eagle's medium (Gibco) supplemented with 15% ES cell FBS (Gibco), 1% non-essential amino acids, 1% l -glutamine, 1% penicillin/streptomycin/Amphotericin B, 0.1% ESGRO-LIF and 0.008% beta-mercaptoethanol.

Techniques: Binding Assay, Functional Assay