293wt cas9 negative control cell lines  (Addgene inc)

Journal: Cell reports

Article Title: SMYD5 is a regulator of the mild hypothermia response

doi: 10.1016/j.celrep.2024.114554

Figure Lengend Snippet: (A) Overview of the genome-scale CRISPR-Cas9 KO approach for the HEK293WT+Cas9+SP1 cell line. (B) Fluorescence measurements and sort gates of the 4 replicates of transduced HEK293WT+Cas9+SP1 cells (green), negative control (HEK293WT, black) and positive control (HEK293WT+Cas9+SP1, gray). (C and D) Genes marked in red are transcription regulators that have a −log10(RRA) score >3.5 and a known repressive function. Genes marked in green are transcription regulators that have a known activating function and a −log10(RRA) score >3.5. Colored dots represent genes that have a −log10(RRA) score >3.5. Orange dots indicate genes that have a positive log fold change (LFC), and blue dots indicate genes that have a negative LFC, from either the SP1 activator (C) or repressor (D) screen. −Log10(RRA) score and LFC were calculated with MAGeCK.

Article Snippet: Using the 293WT + Cas9+SP1 and 293WT + Cas9 (negative control) cell lines we performed a genome wide CRISPR-Cas9 knockout screen (GeCKO) screen, where we transduced the sgRNA pool made from library A and B (Addgene, #1000000049) in MOI 0.3.

Techniques: CRISPR, Fluorescence, Negative Control, Positive Control

Journal: Cell reports

Article Title: SMYD5 is a regulator of the mild hypothermia response

doi: 10.1016/j.celrep.2024.114554

Figure Lengend Snippet: (A) Overexpressed FLAG-tagged SMYD5 ( n = 2) in mESCs binds at promoters of Sp1 and Cirbp but not of Rbm3 . H3K36me3 peaks over Sp1 , Cirbp , and Rbm3 promoter and gene body regions. Data (GEO: GSE184894) are from Zhang et al. (B) Venn graph showing both up- and downregulated SMYD5-bound genes in an RNA-seq of Smyd5 KO cells ( n = 2). Statistical analysis was done with the GeneOverlap package in R using Fisher’s exact test. (C) Smyd5 KO leads to increased mRNA expression of SP1 but not RBM3 or CIRBP in mESCs. (D) SMYD5 knock down (KD) by siRNA yields higher levels of fluorescence of SP1-MHI at 32°C and 37°C compared to the empty vector control. Each data point is a biological replicate ( n = 3) that has been normalized against the same non-transfected HEK293WT+Cas9+SP1-MHI cell line; mean and SD are depicted where applicable. Significance levels were calculated with Šidák’s multiple-comparisons test in GraphPad Prism. (E) Relative expression of SMYD5 mRNA compared to GAPDH from SMYD5 KO cells, measured by RT-qPCR at 37°C, with or without rescue with the FLAG-SMYD5 sgRNAres plasmid (labeled SMYD5 sgRNA#6res). Each data point is a biological replicate ( n = 4), with mean and SD where applicable. Significance levels were calculated with an unpaired one-tailed t test in GraphPad Prism. (F) Western blot using antibodies against SMYD5 and Lamin B in an SMYD5 KO HEK293 cell line at 37°C with and without rescue with the FLAG-SMYD5 sgRNAres plasmid (labeled SMYD5 sgRNA#6res). SMYD5 KO was successful at the protein level at 37°C ( n = 2–3). Data are shown as in (E). (G) Relative expression of SP1 mRNA compared to GAPDH from SMYD5 KO cells, measured by RT-qPCR at 37°C, with or without rescue with the FLAG-SMYD5 sgRNAres plasmid (labeled SMYD5 sgRNA#6res) ( n = 4). Data are shown as in (E). (H–J) Western blot quantification with and without 16-h incubation at 32°C and representative examples using antibodies against SP1, CIRBP, and RBM3, respectively, in SMYD5 KO cells. Each data point is a biological replicate ( n = 2–3), with mean and SD where applicable. Significance levels were calculated with an unpaired one-tailed t test in GraphPad Prism. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Using the 293WT + Cas9+SP1 and 293WT + Cas9 (negative control) cell lines we performed a genome wide CRISPR-Cas9 knockout screen (GeCKO) screen, where we transduced the sgRNA pool made from library A and B (Addgene, #1000000049) in MOI 0.3.

Techniques: RNA Sequencing, Expressing, Knockdown, Fluorescence, Plasmid Preparation, Control, Transfection, Quantitative RT-PCR, Labeling, One-tailed Test, Western Blot, Incubation

Journal: Cell reports

Article Title: SMYD5 is a regulator of the mild hypothermia response

doi: 10.1016/j.celrep.2024.114554

Figure Lengend Snippet:

Article Snippet: Using the 293WT + Cas9+SP1 and 293WT + Cas9 (negative control) cell lines we performed a genome wide CRISPR-Cas9 knockout screen (GeCKO) screen, where we transduced the sgRNA pool made from library A and B (Addgene, #1000000049) in MOI 0.3.

Techniques: Mutagenesis, Inhibition, Reverse Transcription, BIA-KA, Sample Prep, CRISPR, Recombinant, Plasmid Preparation, Software