Journal: The Journal of Experimental Medicine

Article Title: Life-threatening influenza pneumonitis in a child with inherited IRF9 deficiency

doi: 10.1084/jem.20180628

Figure Lengend Snippet: A private IRF9 variant alters mRNA splicing in a child with severe influenza pneumonitis. (A) Pedigrees of the IRF9-deficient family. The double lines connecting the parents indicate consanguinity. The proband is indicated by an arrow. Filled shapes indicate affected individuals while open shapes identify unaffected individuals. (B) Chromatograms demonstrating c.991G>A mutation in patient PBMC-derived DNA (red arrow). (C) Population genetics of homozygous coding missense and predicted loss-of-function IRF9 mutations taken from GnomAD and in-house cohorts. The patient’s variant is private and shown in red, while two other variants, shown in blue, were also identified in our cohort. (D) Schematic illustration of the IRF9 gene. The exons are numbered 1–9, and regions corresponding to functionally significant domains are colored brown (for the DNA-binding domain, DBD), gray (nuclear localization sequence, NLS), or purple (IAD). Patient mutation indicated in red; other mutations indicated in blue. (E) IRF9 transcripts (left panel) and relative frequencies (right panel) produced during exon trapping in U2A cells. The results are representative of two independent experiments. (F) cDNA sequencing to detect the splicing of IRF9 mRNA from F-SV40 cells. Numbers of total and abnormal clones sequenced are indicated. Results representative of two experiments.

Article Snippet: Messenger RNAs were quantified with IRF9 probes Hs00960976-m1 (exon 1–2) and Hs00196051-m1 (exon 7–8; Thermo Fischer Scientific) by qRT-PCR using the Taqman Gene Expression Assay (Applied Biosystems) and normalized to the expression level of HPRT1.

Techniques: Variant Assay, Mutagenesis, Derivative Assay, Binding Assay, Sequencing, Produced, Clone Assay

Journal: The Journal of Experimental Medicine

Article Title: Life-threatening influenza pneumonitis in a child with inherited IRF9 deficiency

doi: 10.1084/jem.20180628

Figure Lengend Snippet: Impact of IRF9 Δex7 on IFN receptor-proximal signaling. (A) qRT-PCR measuring of IRF9 mRNA levels in PBMCs from the patient, her mother, and a healthy control with two probes—one probe spanning intron 7, and a second probe spanning intron 1. Representative results of four independent experiments are shown. (B) Top: WB of endogenous IRF9 in patient F-SV40 cells; GAPDH was used as a loading control. Bottom: STAT and phospho-STAT (pSTAT) levels were also assessed following stimulation with 1,000 U/ml of either IFN–α2b or –γ for 0.5 h on F-SV40 cells from two healthy controls (C1 and C2), the IRF9-deficient patient (IRF9 −/− ), her mother (IRF9 +/− ), a STAT1-deficient patient (STAT1 −/− ), a STAT2-deficient patient (STAT2 −/− ), an IFNGR2-deficient patient (IFNGR2 −/− ), and an IRF7-deficient patient (IRF7 −/− ). Representative results of five independent experiments are shown. (C) WB of IRF9 in IRF9-deficient U2A cells stably transfected with indicated variants (green: variants reported to be loss-of-function in in vitro assays, blue: variants found in-house, red: patient). GAPDH was used as loading control. Representative results of four independent experiments are shown. (D) WB of IRF9 in patient F-SV40 cells stably transfected with indicated variants. GAPDH was used as loading control. Representative results of four independent experiments are shown. (E) WB analysis of IRF9 localization in F-SV40 cells from two healthy controls (C1 and C2), the IRF9-deficient patient (IRF9 −/− ), her mother (IRF9 +/− ), a STAT1-deficient patient (STAT1 −/− ), a STAT2-deficient patient (STAT2 −/− ), an IFNGR2-deficient patient (IFNGR2 −/− ), and an IRF7-deficient patient (IRF7 −/− ). GAPDH and LaminA/C were used as loading controls. Representative results of three independent experiments are shown. (F) Reporter assays of ISRE or GAS-dependent firefly luciferase tested in U2A cells stimulated with 1,000 U/ml of either IFN-α2b or -γ for 16 h after being stably transfected with indicated variants (green: variants reported to be loss-of-function in in vitro assays, blue: variants found in-house, red: patient). The specific response to IFN stimulation was calculated by the ratio of firefly luciferase reporter gene activity to constitutively expressed renilla luciferase activity (RLU, relative luciferase ratio). Representative results of three independent experiments are shown. (G) EMSA analysis of ISRE and GAS binding by IFN-stimulated B-LCLs from three healthy controls (C1, C2, and C3), the IRF9-deficient patient (IRF9 −/− ), her mother (IRF9 +/− ), a STAT1-deficient patient (STAT1 −/− ), a STAT2-deficient patient (STAT2 −/− ), and an IRF7-deficient patient (IRF7 −/− ). Representative results of three independent experiments are shown.

Article Snippet: Messenger RNAs were quantified with IRF9 probes Hs00960976-m1 (exon 1–2) and Hs00196051-m1 (exon 7–8; Thermo Fischer Scientific) by qRT-PCR using the Taqman Gene Expression Assay (Applied Biosystems) and normalized to the expression level of HPRT1.

Techniques: Quantitative RT-PCR, Control, Stable Transfection, Transfection, In Vitro, Luciferase, Activity Assay, Binding Assay

Journal: The Journal of Experimental Medicine

Article Title: Life-threatening influenza pneumonitis in a child with inherited IRF9 deficiency

doi: 10.1084/jem.20180628

Figure Lengend Snippet: Impaired ISG induction in IRF9-deficient cells. (A) Transcription levels of MX1 , IFIT1 , IFIT3 , and CXCL9 assessed by qRT-PCR on F-SV40 cells treated with 1,000 U/ml of IFN–α2b, -β, or –γ for 2 h. Cells were from three healthy controls (C1, C2, and C3), an IRF9-deficient patient (IRF9 −/− ), her mother (IRF9 +/− ), and STAT1-deficient (STAT1 −/− ), STAT2-deficient (STAT2 −/− ), IRF7-deficient (IRF7 −/− ), and IFNGR1-deficient (IFNGR1 −/− ) patients. Representative results of four independent experiments are shown. (B and C) WB of MX1 and IFIT3 on F-SV40 (B) or B-LCL (C) cells treated with 1,000 U/ml of IFN–α2b for various time points. GAPDH was used as a loading control. Representative results of three independent experiments are shown. (D) Transcription levels of MX1 , IFIT1 , IFIT3 , and CXCL9 assessed by qRT-PCR of B-LCL cells treated with 1,000 U/ml of IFN–α2b, -β, or –γ for 2 h. Cells were from three healthy controls (T1, T2, and T3), an IRF9-deficient patient (IRF9 −/− ), her mother (IRF9 +/− ), and STAT1-deficient (STAT1 −/− ), STAT2-deficient (STAT2 −/− ), IRF7-deficient (IRF7 −/− ), and IFNGR2-deficient (IFNGR2 −/− ) patients. Representative results four independent experiments are shown. (E) Transcription levels of MX1 , IFIT1 , and CXCL9 assessed by qRT-PCR in F-SV40 cells from a healthy control (C1), P’s mother (IRF9 +/− ), and P (IRF9 −/− ) stably transfected with luciferase as a control (Luc) or indicated IRF9 variants (WT: WT IRF9, green: reported loss-of-function variants, blue: variants found in-house, red: patient variant). Cells were stimulated with 1,000 U/ml of IFN-α2b, -β, or -γ for 2 or 8 h. Representative results of four independent experiments are shown. (F) Similar to E, qRT-PCR analysis of MX1 , IFIT1 , and CXCL9 expression in parental HT1080 cells and U2A cells. Cells were stimulated with 1,000 U/ml of IFN-α2b, -β, or -γ for 2 or 8 h. Representative results of three independent experiments are shown.

Article Snippet: Messenger RNAs were quantified with IRF9 probes Hs00960976-m1 (exon 1–2) and Hs00196051-m1 (exon 7–8; Thermo Fischer Scientific) by qRT-PCR using the Taqman Gene Expression Assay (Applied Biosystems) and normalized to the expression level of HPRT1.

Techniques: Quantitative RT-PCR, Control, Stable Transfection, Transfection, Luciferase, Variant Assay, Expressing

Journal: The Journal of Experimental Medicine

Article Title: Life-threatening influenza pneumonitis in a child with inherited IRF9 deficiency

doi: 10.1084/jem.20180628

Figure Lengend Snippet: Transcriptomic analysis of ISGs in IRF9-deficient cells. mRNA-seq analysis of primary fibroblasts (A and C) and B-LCLs (B and D) from three healthy controls (C1, C2, and C3), the IRF9-deficient patient (IRF9 −/− ). Cells were treated with 1,000 U/ml IFN–α2b for 2 h. Heatmaps (A and B) show log2 FC values of all ISGs that were found to be differentially regulated (≥1.5-fold) in all three control subjects relative to unstimulated cells. Bar graphs (C and D) quantify the number of ISGs that were differentially regulated (≥1.5-fold) compared with unstimulated cells in healthy controls or the IRF9-deficient patient. (E) Shown are Δ log2 fold change values of a subset of ISGs that were found to be induced ≥1.5-fold (linear scale) in B-LCL cells (upper panels) or primary fibroblasts (lower panels) of the IRF9-deficient patient upon in vitro stimulation with IFN-α. To select this subset of ISGs, the IFN-α2b–induced genes in the healthy controls identified in the mRNA-seq analysis were used. In the IRF9-deficient patient, these genes were first passed through a filter by querying the gene identifiers against the interferome database and by retaining genes that were responsive to in vitro IFN stimulation. ISGs that failed to be induced at least 1.5-fold (linear scale) in patient cells were excluded. The retained ISGs were stratified in three groups of less (Δ less than −0.585), similar (−0.585 < Δ < 0.585), and higher (Δ > 0.585) induced genes relative to the average responses in the healthy control subjects. The numbers of genes in each group are shown in brackets. ***, significant differences at P < 0.0001 by the Kruskal-Wallis test. (F) Log2 FC of induced ISGs in IRF9-deficient B-LCLs (upper panels) or primary fibroblasts (lower panels) and their corresponding values in healthy donors. (G) Network analysis of a subset of highly inducible (> fivefold linear scale) ISGs among control subjects’ B-LCLs and their responsiveness in the IRF9-deficient patient. Biological pathway and physical interactions are depicted as blue and red lines, respectively. 1.5 FC was used as the cut-off to distinguish responsive (red circle) and nonresponsive (blue circle) ISGs. The highly inducible ISGs that were used for query are shaded in yellow.

Article Snippet: Messenger RNAs were quantified with IRF9 probes Hs00960976-m1 (exon 1–2) and Hs00196051-m1 (exon 7–8; Thermo Fischer Scientific) by qRT-PCR using the Taqman Gene Expression Assay (Applied Biosystems) and normalized to the expression level of HPRT1.

Techniques: Control, In Vitro

Journal: The Journal of Experimental Medicine

Article Title: Life-threatening influenza pneumonitis in a child with inherited IRF9 deficiency

doi: 10.1084/jem.20180628

Figure Lengend Snippet: Crippled control of IAV and other viral infections in IRF9-deficient cells. (A) IAV titers in F-SV40 unstimulated (left) or pretreated (right) with 1,000 U/ml IFN-α2 for 16 h, followed by infection with (A/H1N1/CA/2009) IAV at MOI = 1. Mean ± SD ( n = 3) is shown. Cells from three healthy controls were included (C1, C2, and C3), as well as those from the IRF9-deficient patient (IRF9 −/− ), her mother (IRF9 +/− ), and STAT1-deficient (STAT1 −/− ), STAT2-deficient (STAT2 −/− ), and IRF7-deficient (IRF7 −/− ) patients. Four independent experiments (mean ± SD) are shown. (B) VSV titers in F-SV40 cells unstimulated (left) or pretreated (right) with 1,000 U/ml IFN-α2 for 16 h, followed by infection with VSV at MOI = 3. Four independent experiments (mean ± SD) are shown. (C) IAV titers in stably transfected F-SV40 cells unstimulated (left) or pretreated (right) with 1,000 U/ml IFN-α2 for 16 h, followed by infection with IAV at MOI = 1. Cells were from a healthy control (C1), a STAT1-deficient patient (STAT1 −/− ), P (IRF9 −/− ), and P’s cells stably transfected with luciferase or WT IRF9 (gray), variants reported to be loss-of-function in in vitro assays (green), variants found in-house (blue), or the patient’s variant (red). Three independent experiments (mean ± SD) are shown. (D) VSV titers in stably transfected F-SV40 cells unstimulated (left) or pretreated (right) with 1,000 U/ml IFN-α2 for 16 h, followed by infection with VSV at MOI = 3. Four independent experiments (mean ± SD) are shown. (E) Percentage of RSV-infected (GFP + ) F-SV40 cells at 24 and 48 h after infection. Cells from three healthy controls were included (C1, C4, and C5, black), as well as those from the IRF9-deficient patient (IRF9 −/− , red), and cells from STAT1-deficient (STAT1 −/− ), STAT2-deficient (STAT2 −/− ), and IRF7-deficient (IRF7 −/− ) patients. Three independent experiments (mean ± SD) are shown. (F) Mean fluorescence intensity (MFI) of RSV-infected (GFP + ) F-SV40 cells at 24 and 48 h after infection. Three independent experiments (mean ± SD) are shown. (G) Percentage of PIV-infected (GFP + ) F-SV40 cells at 24 and 48 h after infection. Three independent experiments (mean ± SD) are shown. (H) MFI of PIV-infected (GFP + ) F-SV40 cells at 24 and 48 h after infection. Three independent experiments (mean ± SD) are shown. MFI of GFP + cells in individual samples were normalized to the averaged MFI of the three healthy controls at 24 h after infection in F and H.

Article Snippet: Messenger RNAs were quantified with IRF9 probes Hs00960976-m1 (exon 1–2) and Hs00196051-m1 (exon 7–8; Thermo Fischer Scientific) by qRT-PCR using the Taqman Gene Expression Assay (Applied Biosystems) and normalized to the expression level of HPRT1.

Techniques: Control, Infection, Stable Transfection, Transfection, Luciferase, In Vitro, Variant Assay, Fluorescence

Journal: The Journal of Experimental Medicine

Article Title: Life-threatening influenza pneumonitis in a child with inherited IRF9 deficiency

doi: 10.1084/jem.20180628

Figure Lengend Snippet: IRF9 is required for optimal control of viral infections . (A) WB confirms the efficiency of RNAi of IRF9 or MAVS in primary dermal fibroblasts. (B–F) Primary human dermal fibroblasts previously transfected with the indicated siRNA (negative control, IRF9, MAVS) were tested for control of HRV, RSV, and PIV. Cells were infected with HRV-A16 at MOI of 10 (B), RSV at MOI of 0.5 (C and D), or PIV3 at MOI of 0.1 (E and F). Relative HRV transcripts (B) were measured by qRT-PCR, and values were normalized to the siNeg control. Percentage of infected cells (C and E) and relative virus per infected cell (D and F) were measured by flow cytometric analysis of GFP + cells. MFI of GFP + cells in individual samples were normalized to negative control at 24 h (D and F). Shown are the mean ± SD of six (B–F) experiments. *, P < 0.05; **, P < 0.01, by Kruskal-Wallis test.

Article Snippet: Messenger RNAs were quantified with IRF9 probes Hs00960976-m1 (exon 1–2) and Hs00196051-m1 (exon 7–8; Thermo Fischer Scientific) by qRT-PCR using the Taqman Gene Expression Assay (Applied Biosystems) and normalized to the expression level of HPRT1.

Techniques: Control, Transfection, Negative Control, Infection, Quantitative RT-PCR, Virus

Journal: Nature Communications

Article Title: Pan-cancer transcriptomic analysis associates long non-coding RNAs with key mutational driver events

doi: 10.1038/ncomms13197

Figure Lengend Snippet: ( a ) Total RNA sequencing of A549 lung cancer cells treated with NFE2L2 siRNAs ( n =3 transfections using three unique sequences) or control siRNAs ( n =4 transfections using two unique sequences) during 48 h. Left panel: volcano plot showing 56 lncRNAs (black dots) altered at q <0.01 (DESeq2), with aggregation of predicted NFE2L2- responsive lncRNAs (red circles) among top repressed transcripts. Right panel: predicted NFE2L2 -responsive lncRNAs are repressed 48 h post NFE2L2 inhibition. The x -axis shows expression ratios in NFE2L2 mutated compared with wild type tumours for cancer types with association P <0.001 (Wilcoxon rank sum test). Vertical bars indicate, for each gene, the mean log 2 ratio across the relevant cancer types and coloured dots show the individual cancers using colours from . The plot includes 11/15 predicted lncRNAs detectable in A549 cells (≥10 reads in one sample). ( b ) Validation of select transcripts by RT-qPCR, comparing cells treated with NFE2L2 siRNAs or control siRNA as described in a , with additional results from H838 lung cancer cells. Values were normalized to ACTB, and are shown relative to the controls. P -values were determined using Student's t -test. ( c ) Induction of LINC00942 , RP11-284F21.7 and RP11-345L23.1 in NFE2L2 mutated compared with wild type tumours (cancers with P <0.05 are shown, Wilcoxon rank sum test). Genomic contexts are shown; blue, lncRNAs; green, coding genes. * P <0.05; ** P <0.001; *** P <1e-4. ( d ) Induction of LINC00942 , RP11-284F21.7 and RP11-345L23.1 in KEAP1 mutated compared with wild type tumours. Error bars indicate s.e.m.

Article Snippet: RNA was prepared using the RNeasy Mini kit (Qiagen), and cDNA synthesized with DyNAmo cDNA Synthesis kit (Thermo Scientific) and gene expression levels determined using TaqMan assays (Life Technologies; ACTB , Hs01060665_g1; NFE2L2 , Hs00975961_g1; LINC00942 , Hs03669859_m1; RP11-284F21.7 , custom assay targeting exon 1–2 junction; RP11-345L23.1, custom assay targeting exon 2–3 junction; GCLC , Hs00155249_m1; TXNRD1 , Hs00917067_m1; NQO1 , Hs00168547_m1; BCAN , Hs00222607_m1) as previously described .

Techniques: RNA Sequencing, Transfection, Control, Inhibition, Expressing, Biomarker Discovery, Quantitative RT-PCR

Journal: Nature Communications

Article Title: Pan-cancer transcriptomic analysis associates long non-coding RNAs with key mutational driver events

doi: 10.1038/ncomms13197

Figure Lengend Snippet: ( a ) Cells were treated with three different antisense oligos (ASOs) targeting LINC00942 (942-ASO1-3, n =2 transfections each), control LNA oligos (Ctrl-ASO1-2, n =2 transfections each), or vehicle only (Mock, n =3 transfections) during 36 h. RT-qPCR-based expression values were normalized to ACTB, and are shown relative to the mean of the control samples. P -values from Student's t -test using the four control ASOs transfections as reference. ( b ) Transfection with ASOs complementary to LINC00942 led to reduced expression of GCLC , required for glutathione synthesis. ( c ) GCLC western blot with GAPDH shown as control (36 h post transfection). ( d ) Reduced glutathione (GSH) levels 72 h post transfection with LINC00942 ASOs in A549 cells, as determined by the GSH-Glo assay ( n =3 transfections per group). P -values from Student's t -test using the six control ASO transfections as reference. ( e ) Increased levels of reactive oxygen species 72 h post transfection with LINC00942 ASOs in A549 cells, as determined by flow cytometric 2′,7′-dichlorodihydrofluorescein (DCF) assays ( n =3 transfections per group). P -values from Student's t -test using the six control ASO transfections as reference. Error bars indicate s.e.m.

Article Snippet: RNA was prepared using the RNeasy Mini kit (Qiagen), and cDNA synthesized with DyNAmo cDNA Synthesis kit (Thermo Scientific) and gene expression levels determined using TaqMan assays (Life Technologies; ACTB , Hs01060665_g1; NFE2L2 , Hs00975961_g1; LINC00942 , Hs03669859_m1; RP11-284F21.7 , custom assay targeting exon 1–2 junction; RP11-345L23.1, custom assay targeting exon 2–3 junction; GCLC , Hs00155249_m1; TXNRD1 , Hs00917067_m1; NQO1 , Hs00168547_m1; BCAN , Hs00222607_m1) as previously described .

Techniques: Transfection, Control, Quantitative RT-PCR, Expressing, Western Blot, Glo Assay

Journal: Heliyon

Article Title: The association between mannose binding lectin gene polymorphisms and the risk of neonatal sepsis: an updated meta-analysis

doi: 10.1016/j.heliyon.2023.e14905

Figure Lengend Snippet: Characteristics of 15 studies included in the meta-analysis.

Article Snippet: MBL exon1 gene polymorphism , , , , , , .

Techniques: Nested PCR

Journal: Heliyon

Article Title: The association between mannose binding lectin gene polymorphisms and the risk of neonatal sepsis: an updated meta-analysis

doi: 10.1016/j.heliyon.2023.e14905

Figure Lengend Snippet: Gene distribution of 15 studies included in the meta-analysis.

Article Snippet: MBL exon1 gene polymorphism , , , , , , .

Techniques: Variant Assay

Journal: Heliyon

Article Title: The association between mannose binding lectin gene polymorphisms and the risk of neonatal sepsis: an updated meta-analysis

doi: 10.1016/j.heliyon.2023.e14905

Figure Lengend Snippet: Meta-analysis of the association between MBL gene polymorphism and NS under different gene models.

Article Snippet: MBL exon1 gene polymorphism , , , , , , .

Techniques:

Journal: Heliyon

Article Title: The association between mannose binding lectin gene polymorphisms and the risk of neonatal sepsis: an updated meta-analysis

doi: 10.1016/j.heliyon.2023.e14905

Figure Lengend Snippet: Meta-analysis of association between MBL gene polymorphism and neonatal sepsis under variant type vs. wild type. (A) MBL exon1 gene ploymorphism (AO + OO). (B) rs1800450. (C) rs1800451. (D) rs5030737.

Article Snippet: MBL exon1 gene polymorphism , , , , , , .

Techniques: Variant Assay

Journal: Molecular cell

Article Title: PRC2.1 and PRC2.2 Synergize to Coordinate H3K27 Trimethylation.

doi: 10.1016/j.molcel.2019.08.012

Figure Lengend Snippet: Figure 1. PRC2.1 and PRC2.2 Co-occupy the Majority of Sites in ESCs (A) Composition of PRC2.1 and PRC2.2. (B) Average ChIP-Rx signal profiles for SUZ12, H3K27me3, and PRC2.1- (MTF2/PCL2 and EPOP) and PRC2.2- (JARID2 and AEBP2) specific accessory proteins in wild-type (WT) ESCs. (C) Heatmap representation of PRC2.1 only and PRC2.1/2.2 shared regions. Plots are centered on region midpoint ±2.5 kb. Relative intensities are indicated. (D) Genome browser representations of ChIP-Rx normalized reads for MTF2/PCL2, EPOP, JARID2, AEBP2, SUZ12, and H3K27me3 at representative PRC2.1/ PRC2.2 shared and PRC2.1 only genomic sites in WT ESCs.

Article Snippet: Generation of Jarid2 single and Pcl1-3/Jarid2 quadruple knock-out cell lines Pcl1-3/Jarid2 quadruple knock out ESCs (QKO) were generated by transfecting TKO ESCs with 2 pSpCas9 (BB)-2A-eGFP vectors (Addgene, px458) containing gRNAs targeting each side of Jarid2 Exon 1, using Lipofectamine 2000 as per manufacturer’s recommendations.

Techniques:

Journal: Molecular cell

Article Title: PRC2.1 and PRC2.2 Synergize to Coordinate H3K27 Trimethylation.

doi: 10.1016/j.molcel.2019.08.012

Figure Lengend Snippet: Figure 7. The Combined Loss of PRC2.1 and PRC2.2 Leads to Global Redistribution of SUZ12 (A) Top: schematic illustrating the generation of Pcl1–3/Jarid2 quadruple KO (QKO) ESCs. Bottom: western blot analyses using the indicated antibodies on whole- cell lysates from TKO, two independent QKO clones, and matched WT ESCs. (B) Western blot analyses using the indicated antibodies on nuclear lysates from two independent QKO ESC lines (c1 and c2) and matched WT ESCs. (C) Quantitative ChIP analyses using the indicated antibodies at a selection of broad and narrow Polycomb sites from TKO, QKO (c1), and matched WT ESCs. All experiments were performed at least three times and a representative experiment is shown. Error bars are representative of technical triplicates. (D) Genome browser representations of ChIP-Rx normalized reads of SUZ12, JARID2, EPOP, AEBP2, and RYBP at representative, narrow (Prmt8), and broad (HoxA) loci in QKO (c1) and matched WT ESCs. (E) Average ChIP-Rx signal profiles of JARID2, SUZ12, EPOP, CBX7, and RYBP at their individual peak sets (±20 kb) in TKO, QKO (c1), and matched WT ESCs.

Article Snippet: Generation of Jarid2 single and Pcl1-3/Jarid2 quadruple knock-out cell lines Pcl1-3/Jarid2 quadruple knock out ESCs (QKO) were generated by transfecting TKO ESCs with 2 pSpCas9 (BB)-2A-eGFP vectors (Addgene, px458) containing gRNAs targeting each side of Jarid2 Exon 1, using Lipofectamine 2000 as per manufacturer’s recommendations.

Techniques: Western Blot, Clone Assay, Selection