Journal: Nucleic Acids Research

Article Title: Cytoplasmic DNA can be detected by RNA fluorescence in situ hybridization

doi: 10.1093/nar/gkz645

Figure Lengend Snippet: Different plasmids containing non-expressed genes are also detected in cytoplasmic puncta after transient transfection. U2OS Cells were transiently transfected overnight by PolyJet with constructs containing ( A ) the cyclin D1 ( CCND1 ) gene without a promoter, ( B ) the sequence of the MEG3 lncRNA gene, without a promoter, and ( C ) only MS2 sequences without a promoter or a coding region. In all RNA FISH experiments the cells showed cytoplasmic puncta (yellow) with specific probe sets to each gene, and were still detected after actinomycin D (middle) or RNase (bottom) treatments. Hoechst DNA counterstain is in cyan. DIC is in grey. Scale bar = 10 μm.

Article Snippet: The plasmid containing MS2 repeats only (pSL-24xMS2, Addgene #31865) does not contain a promoter ( ).

Techniques: Transfection, Construct, Sequencing

Journal: Nucleic Acids Research

Article Title: Cytoplasmic DNA can be detected by RNA fluorescence in situ hybridization

doi: 10.1093/nar/gkz645

Figure Lengend Snippet: Two different probe sets to the same plasmid colocalize in cytoplasmic puncta detected by RNA FISH. ( A ) U2OS cells were transfected overnight with a plasmid containing the cyclin D1 ( CCND1 ) gene and MS2 repeats in the 3′UTR, and no promoter, using PolyJet. RNA FISH was performed with two different probe sets to the cyclin D1 (yellow) and MS2 (cyan) regions. Colocalization between the cytoplasmic puncta can be seen in the merge. Actinomycin D treatment did not abolish the cytoplasmic dots (bottom). ( B ) U2OS Tet-on cells were transfected overnight with a plasmid containing the E6 gene that encodes a CFP fusion protein and has MS2 repeats in the 3′UTR. RNA FISH was performed with two different probe sets to the MS2 (yellow) and CFP (cyan) regions. Colocalization between the cytoplasmic puncta can be seen in the merge. The detection of the cytoplasmic puncta was not affected when the E6 mRNA was transcribed after induction by doxycycline (dox). Arrowheads point to a cell containing E6 mRNA. ( C ) U2OS cells were transfected overnight with a plasmid containing the PANDA gene on the antisense strand and the cyclin D1 gene and MS2 sequences on the sense strand. There was colocalization between the cytoplasmic puncta detected with the PANDA (yellow) and MS2 (cyan) FISH probes. Actinomycin D treatment did not abolish the cytoplasmic dots (bottom). Hoechst DNA counterstain is in cyan. Scale bars = 10 μm.

Article Snippet: The plasmid containing MS2 repeats only (pSL-24xMS2, Addgene #31865) does not contain a promoter ( ).

Techniques: Plasmid Preparation, Transfection

Journal: bioRxiv

Article Title: A single-cell transcriptomics CRISPR-activation screen identifies new epigenetic regulators of zygotic genome activation

doi: 10.1101/741371

Figure Lengend Snippet: A) Scatterplot showing normalised expression levels (log 2 reads per million; RPM) of SAM ESCs against their parental cell line E14, analysed by RNA-sequencing, highlighting dCas9-VP64 and MS2-p65-HSF1 transcripts in black and screening candidates in blue. B) Histograms showing number of unique molecular identifiers (UMIs) for each analysed sample; cells within the red bars (15,000<40,000 UMIs) were retained as quality control (QC)-passing cells. C) Histograms showing number of detected genes for each analysed sample; cells within the red bars (4,000<6,500 genes) were retained as QC-passing cells. D) Histograms showing the percentage of reads from mitochondrial reads for each analysed sample; cells below the red bars (<5%) were retained as QC-passing cells. E, F) Dot-plot (upper panels) and density representation (lower panels) of MERVL (E) and Zscan4b/c/d/e/f (sum of all genes) (F) log-normalised expression levels in counts per million in untransduced E14 ESCs (grey), SAM ESCs transduced with a non-targeting sgRNA (blue), SAM ESCs transduced with sgRNAs targeting MERVL LTRs (pink) and SAM ESCs transduced sgRNAs targeting Zscan4 gene promoters (orange), analysed by 10X Genomics 3’ single-cell RNA-sequencing. Reported percentages were calculated with the cells above the vertical black line from the total number of cells in each sample. G) Dot-plot (upper panels) and density representation (lower panels) of ZGA genes (as described in Table S2) log-normalised expression in counts per million (sum of all genes) in the samples described in E) and F) . Reported percentages were calculated with the cells above the vertical black line from the total number of cells in each sample. H) Dot-plot showing normalised expression levels (log 2 reads per million; RPM) of the screening candidates in oocytes, zygotes, 2-cell and 4-cell embryos. Data analysed from ). I) Schematic representation of CROP-sgRNA-MS2 lentiviral vector and transcripts produced upon lentiviral reverse transcription and integration in the host genome. J) Histogram showing normalised log 10 average of read 1 and read 2 from next-generation sequencing analysis of the 475 oligo sgRNA library cloned into the lentiviral backbone described in I) ; representation of 90% of sgRNAs in the library is within 19.9 folds.

Article Snippet: Next, they were cotransfected with 3.5 μg of pMD2.G (Addgene 12259), 6.5 μg of psPAX2 (Addgene 12260) and 10μg of the lentiviral vector of interest: dCas9-VP64_Blast (Addgene 61425), MS2-p65-HSF1_Hygro (Addgene 61426), sgRNA(MS2)_puro (Addgene 73795) cloned with an individual sgRNA, or CROP-sgRNA-MS2 cloned with an individual sgRNA.

Techniques: Expressing, RNA Sequencing Assay, Transduction, Plasmid Preparation, Produced, Next-Generation Sequencing, Clone Assay

Journal: eLife

Article Title: BAP1/ASXL complex modulation regulates epithelial-mesenchymal transition during trophoblast differentiation and invasion

doi: 10.7554/eLife.63254

Figure Lengend Snippet:

Article Snippet: Recombinant DNA reagent , Lenti MS2-p65-HSF1-Hygro (plasmid) , Addgene , RRID: Addgene_61426 , Lentiviral plasmid to transfect HEK293T cells and package MS2-p65-HSF1 viral particles.

Techniques: Recombinant, Plasmid Preparation, Expressing, Clone Assay, CRISPR, Transduction, Sequencing, Small Interfering RNA, Negative Control, Transfection, Cell Adhesion Assay, Software