lentivirus-expressing human sdc-1 gene or sdc-1 shrna sequence (5’-gcaagatatcaccttgtcaca-3)  (Genechem)

Journal: bioRxiv

Article Title: Genome-wide CRISPR interference screen identifies Clip2 as a novel regulator of osteocyte maturation and morphology

doi: 10.1101/2025.10.23.683957

Figure Lengend Snippet: A) Timeline of shRNA infection and analysis of mRNA and protein expression. B) Western blot shows expression of eIF3B protein in Ocy454 cells at differentiation day 7 following stable infection with the indicated shRNAs. C) mRNA expression of Eif3b in Ocy454 cells at differentiation day 7 following stable infection with the indicated shRNAs. Line shows median of three biologic replicates. *p<0.05. D) Flow cytometry results show percent of cells labeled with CD61-PE antibody compared to isotype control at a range of differentiation timepoints following stable infection with the indicated shRNAs. Error bars show standard deviation for all replicates at each time point. E) mRNA expression of Itgb3 in Ocy454 cells at differentiation day 10 following stable infection with the indicated shRNAs. Line shows median of three biologic replicates. *p<0.05. F) Histograms show intensity of CD61-PE fluorescence compared to isotype control at differentiation day 7 for cells stably expressing FLuc-targeting shRNA and Itgb3-targeting shRNA. G) Histograms show intensity of CD61-PE fluorescence compared to isotype control at differentiation day 7 for cells stably expressing FLuc-targeting shRNA and Itgb3-targeting shRNA.

Article Snippet: CRISPRi sgRNA sequences and shRNA sequences were designed using Broad Institute tools CRISPick ( https://portals.broadinstitute.org/gppx/crispick/public ) and TRC shRNA Design ( https://portals.broadinstitute.org/gpp/public/seq/search ).

Techniques: shRNA, Infection, Expressing, Western Blot, Flow Cytometry, Labeling, Control, Standard Deviation, Fluorescence, Stable Transfection

Journal: bioRxiv

Article Title: Genome-wide CRISPR interference screen identifies Clip2 as a novel regulator of osteocyte maturation and morphology

doi: 10.1101/2025.10.23.683957

Figure Lengend Snippet: A-D) mRNA expression of targeted genes at differentiation day 7 in Ocy454 cells stably expressing the indicated shRNAs. Line shows median of three biologic replicates. *p<0.05 compared to FLuc. E-H) Histograms show intensity of CD61-PE fluorescence compared to isotype control at differentiation day 7 for cells stably expressing FLuc-targeting shRNA and the indicated targeting shRNA for one representative sample. I) Summary of flow cytometry results showing median fluorescence intensity (MFI) of CD61-PE at differentiation day 7 in cells stably expressing the indicated shRNAs. Error bars show standard deviation of all replicates. *p<0.05 compared to FLuc.

Article Snippet: CRISPRi sgRNA sequences and shRNA sequences were designed using Broad Institute tools CRISPick ( https://portals.broadinstitute.org/gppx/crispick/public ) and TRC shRNA Design ( https://portals.broadinstitute.org/gpp/public/seq/search ).

Techniques: Expressing, Stable Transfection, Fluorescence, Control, shRNA, Flow Cytometry, Standard Deviation

Journal: bioRxiv

Article Title: Genome-wide CRISPR interference screen identifies Clip2 as a novel regulator of osteocyte maturation and morphology

doi: 10.1101/2025.10.23.683957

Figure Lengend Snippet: A) mRNA expression of Astn1 in Ocy454 cells at differentiation day 7 following stable infection with the indicated shRNAs. Line shows median of three biologic replicates. B) Histograms show intensity of CD61-PE fluorescence compared to isotype control at differentiation day 7 for cells stably expressing FLuc-targeting shRNA and Astn1-targeting shRNA for one representative sample. C) Summary of flow cytometry results showing median fluorescence intensity (MFI) of CD61-PE at differentiation day 7 in cells stably expressing the indicated shRNAs. Error bars show standard deviation of all replicates. *p<0.05 compared to FLuc. D-L) Histograms show intensity of CD61-PE fluorescence compared to isotype control at differentiation day 7 for cells stably expressing FLuc-targeting shRNA and the indicated targeting shRNA for one representative sample.

Article Snippet: CRISPRi sgRNA sequences and shRNA sequences were designed using Broad Institute tools CRISPick ( https://portals.broadinstitute.org/gppx/crispick/public ) and TRC shRNA Design ( https://portals.broadinstitute.org/gpp/public/seq/search ).

Techniques: Expressing, Infection, Fluorescence, Control, Stable Transfection, shRNA, Flow Cytometry, Standard Deviation

Journal: bioRxiv

Article Title: Genome-wide CRISPR interference screen identifies Clip2 as a novel regulator of osteocyte maturation and morphology

doi: 10.1101/2025.10.23.683957

Figure Lengend Snippet: A-G) mRNA expression of osteocyte maturity genes at differentiation day 10 in Ocy454 cells stably expressing the indicated shRNAs. *p<0.05 compared to FLuc. H) Alpha-tubulin immunofluorescence or isotype control immunofluorescence (red) and DAPI (blue) in Ocy454 cells without shRNA infection. Scale bars = 20 μm. I) Ocy454 cells labeled with phalloidin (green) and DAPI (blue) following stable expression of the indicated shRNAs. Scale bars = 20 μm. J-K) Cell Profiler measurements of form factor and perimeter. Each plotted point represents metrics of one cell. Lines designate the median and interquartile range of n=47-158 cells. *p<0.05.

Article Snippet: CRISPRi sgRNA sequences and shRNA sequences were designed using Broad Institute tools CRISPick ( https://portals.broadinstitute.org/gppx/crispick/public ) and TRC shRNA Design ( https://portals.broadinstitute.org/gpp/public/seq/search ).

Techniques: Expressing, Stable Transfection, Immunofluorescence, Control, shRNA, Infection, Labeling