The animals (2 to 4 months old) bearing both the LCR+β(ε) and β(γ)+3'HS1 knock-in alleles in trans were made anemic and nucleated erythroid cells were collected from their spleens. Following fixation with 1% formaldehyde for 10 min at room temperature. Nuclei (2 x 107 cells) were digested with 12.5 units/ml of micrococcal nuclease at 37°C for 20 min. The chromatin was incubated with anti-CTCF antibody (D31H2; Cell Signaling Technology) or purified rabbit IgG (Invitrogen) overnight at 4°C and was precipitated with preblocked Dynabeads protein G magnetic beads (Life Technologies, Carlsbad, CA). Immunoprecipitated materials were then washed and reverse cross-linked. DNA was purified with the QIAquick PCR purification kit (Qiagen, Venlo, The Netherlands) and subjected to qPCR analysis. The endogenous H19 ICR and Necdin sequences were analyzed as positive and negative controls, respectively [41 (link)]. LCR-HS5 and 3'HS1 primer sets were as follows:
Anti ctcf antibody d31h2
Manufactured by Cell Signaling Technology
The Anti-CTCF antibody (D31H2) is a primary antibody used in research applications. It is designed to specifically recognize and bind to the CTCF protein, which is a transcription factor involved in regulating gene expression and chromatin organization.
Automatically generated - may contain errors
Lab products found in correlation
Purified rabbit igg, by Thermo Fisher Scientific (3 mentions)
Dynabeads protein g magnetic beads, by Thermo Fisher Scientific (3 mentions)
Qiaquick pcr purification kit, by Qiagen (3 mentions)
Anti cd45.1 a20, by BioLegend (1 mentions)
Anti cd44 im7, by Thermo Fisher Scientific (1 mentions)
Anti il 7rα a7r34, by BioLegend (1 mentions)
Anti tcf1 c63d9, by Cell Signaling Technology (1 mentions)
Foxp3 transcription factor staining buffer set, by Thermo Fisher Scientific (1 mentions)
Facsverse, by BD (1 mentions)
Facscelesta, by BD (1 mentions)
4 protocols using anti ctcf antibody d31h2
1
Chromatin Immunoprecipitation of CTCF Binding
2
Characterization of CD8+ T Cell Phenotypes
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Single-cell suspensions were prepared from the spleen, LNs, and surface or intracellularly stained as described (Shan et al., 2022a (link)). The fluorochrome-conjugated antibodies were as follows: anti-CD8 (53–6.7), anti-TCRβ (H57-597), anti-CD45.2 (104), anti-Granzyme B (GB12), anti-IFN-γ (XMG1.2), anti-Tbet (4B10), anti-CD62L (MEL-14), anti-KLRG1 (2F1), anti-CD25 (PC61.5), anti-CD69 (H1.2F3), and anti-CD44 (IM7) were from Thermo Fisher Scientific; anti-Tcf1 (C63D9) from Cell Signaling Technology; anti-IL-7Rα (A7R34) and anti-CD45.1 (A20) were from BioLegend. For detection of Tcf1 and Tbet proteins, surface-stained cells were fixed and permeabilized with the Foxp3/Transcription Factor Staining Buffer Set (eBiosciences, Thermo Fisher Scientific), followed by incubation with corresponding fluorochrome-conjugated antibodies. Apoptotic cells were detected with FLICA 660 Caspase-3/7 detection kit (Bio-Rad). Data were collected on FACSCelesta or FACSVerse (BD Biosciences) and were analyzed with FlowJo software V10.2 (TreeStar). For validation of CTCF deletion efficiency, cell lysates from sorted GFP+ naive or early effector CD8+ T cells were immunoblotted with anti-CTCF antibody (D31H2; Cell Signaling Technology) following standard protocols.
3
CTCF Chromatin Immunoprecipitation in Erythroid Cells
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The LCb118 YAC-TgM (2–4 months old) inheriting the transgene either paternally or maternally were made anaemic by phenylhydrazine treatment, and nucleated erythroid cells were collected from their spleens. Livers were obtained from E18.5 embryos inheriting the LCb118 knock-in allele either paternally or maternally. Cells were fixed in PBS with 1% formaldehyde for 10 min at room temperature. Nuclei (2 × 107 cells) were digested with 12.5 units/ml of micrococcal nuclease at 37 °C for 20 min to prepare primarily mono- to di-nucleosome-sized chromatin. The chromatin was incubated with anti-CTCF antibody (D31H2; Cell Signaling Technology) or purified rabbit IgG (Invitrogen) overnight at 4 °C and was precipitated with preblocked Dynabeads protein G magnetic beads (Life Technologies, Carlsbad, CA). Immunoprecipitated materials were then washed extensively and reverse cross-linked. DNA was purified with the QIAquick PCR purification kit (Qiagen, Venlo, the Netherlands) and subjected to qPCR analysis. The endogenous H19 ICR and Necdin sequences were analyzed as positive and negative controls, respectively [35 (link)]. PCR primers were reported previously [35 (link)].
4
CTCF Binding Site Identification
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The LCb478 YAC-TgM (2–4 months old) inheriting the transgene paternally or maternally were made anemic by phenylhydrazine treatment. Nucleated erythroid cells were collected from their spleens and fixed in PBS with 1% formaldehyde for 10 min at room temperature. Nuclei (2 × 107 cells) were digested with 12.5 units/ml of micrococcal nuclease at 37 °C for 20 min to prepare primarily mono- to di-nucleosome-sized chromatin. The chromatin was incubated with anti-CTCF antibody (D31H2; Cell Signaling Technology) or purified rabbit IgG (Invitrogen) overnight at 4 °C and was precipitated with preblocked Dynabeads protein G magnetic beads (Life Technologies, Carlsbad, CA). Immunoprecipitated materials were then washed extensively and reverse cross-linked. DNA was purified with the QIAquick PCR purification kit (Qiagen, Venlo, the Netherlands) and subjected to qPCR analysis. The endogenous H19 ICR and Necdin sequences were analyzed as positive and negative controls, respectively, [41 (link)]. PCR primers were reported previously [41 (link)].
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