Cells were grown to 80% confluence, washed twice in warm PBS, dissociated with 0.5x Accutase (Catalogue # SCR005; Millipore), and re-suspended in warm medium (DMEM F-12 Catalogue # 11320-033; Invitrogen) containing 0.1 volume crosslinking solution (Kondo, Shen, Yan, Huang & Issa, 2004 (link)). ChIP reactions were performed as described previously (Veazey et al., 2015 (link)) followed by DNA purification with a Qiaquick PCR Cleanup kit (Catalogue # 28106; QIAGEN). Antibodies used include: anti-H3K4me3 (Catalogue # 04-745; Millipore), anti- H3K27me3 (Catalogue # 39155; Active Motif), anti-H3K9ac (Catalogue # 07-352; Millipore), and anti-H3K9me2 (Catalogue # 39239; Active Motif). Antibodies for modified histones were used at 1 μg/ChIP reaction. The concentration of IgG (Catalogue # SC-2027; Santa Cruz) was also used at 1 ug/ChIP reaction. For analysis of candidate loci, real-time PCR was performed with the Dynamo Flash supermix (Catalogue # F-415XL; Thermo Scientific) according to the recommended protocol. Reactions were performed on a Bio-Rad CFX384 Touch PCR system. Data was analyzed using the formula previously described (Mukhopadhyay, Deplancke, Walhout & Tissenbaum, 2008 (link)). Primer sequences are listed in Table S1 - Primer Sequences.
Cfx384 touch pcr system
Manufactured by Bio-Rad
The CFX384 Touch PCR system is a real-time PCR detection system designed for quantitative gene expression analysis. It features a 384-well format, touch screen interface, and compatibility with a wide range of fluorescent dyes and probes.
Automatically generated - may contain errors
Lab products found in correlation
Dmem f12, by Thermo Fisher Scientific (3 mentions)
Anti h3k9me2, by Active Motif (3 mentions)
Dynamo flash supermix, by Thermo Fisher Scientific (3 mentions)
Accutase, by Merck Group (2 mentions)
Anti h3k4me3, by Merck Group (2 mentions)
Anti h3k9ac, by Merck Group (2 mentions)
Anti h3k27me3, by Active Motif (2 mentions)
Qiaquick pcr cleanup kit, by Qiagen (2 mentions)
Allprep dna rna protein mini kit, by Qiagen (1 mentions)
Dynamo flash sybr qpcr kit, by Thermo Fisher Scientific (1 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Qiaquick pcr purification kit, by Qiagen (1 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Anti satb2, by Abcam (1 mentions)
Anti h4k20me3, by Active Motif (1 mentions)
4 protocols using cfx384 touch pcr system
1
Chromatin Immunoprecipitation (ChIP) Assay
2
Fetal Tissue RNA Extraction and qPCR
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We isolated total RNA from the GD17 fetal cerebellum and heart using the AllPrep DNA/RNA/Protein Mini Kit (Catalog No. 80004; Qiagen) according to the manufacturer’s instructions. We then seeded 1 μg of RNA into a reverse transcription reaction using the High Capacity cDNA Reverse Transcription Kit (Catalog No. 4368814; Thermo Fisher) following the recommended protocol. We assayed gene expression using the DyNamo Flash SYBR qPCR kit (Catalog No. F-415; Thermo Fisher) on a Bio-Rad CFX384 Touch PCR system. Primer sequences are listed in Additional file 1 . To normalize gene expression levels, we assayed the expression of the reference genes Glyceraldehyde 3-phosphate dehydrogenasetyrosine (Gapdh), 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta (Ywhaz), and H2A Histone family member Z (H2afz). For studies in the fetal cortex, we used Gapdh and Ywhaz. For studies in the cerebellum and heart, we replaced Gapdh with H2afz due to increased stability [35 (link)].
3
Chromatin Immunoprecipitation from Cortical Tissue
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We thawed the cortices derived from a single brain and filtered the tissues into a single-cell suspension using gentle mechanical dissociation. We washed cells twice with PBS containing protease inhibitor cocktail (Cat# 78437; Thermo Scientific) and re-suspended them in medium (DMEM F-12 Cat# 11320-033; Invitrogen) containing 0.1 volume crosslinking solution [33 (link)]. We carried out chromatin immunoprecipitation reactions following the previously published protocol [34 (link)]. The specific antibodies used in this study include anti-H3K9me2 (Cat# 39239; Active Motif, RRID:AB_2793199), antiH3K9me3 (Cat# 05-1242; Millipore-Sigma, RRID:AB_1587136), antiH4K20me3 (Cat# 91107; Active Motif, RRID:AB_2793777), anti-SATB2 (Cat# ab34735; Abcam, RRID:AB_2301417) and the negative IgG control (Cat# SC-2027; Santa Cruz, RRID:AB_737197). We used antibodies for modified histones and the IgG control at a concentration of 1 µg/ChIP reaction and 4 µg/ChIP reaction for SATB2. We purified precipitated DNA using the QIAquick PCR Purification Kit (catalog # 28106, Qiagen) and assayed the enrichment of the indicated sequences using quantitative PCR. We performed qPCR using the Dynamo Flash supermix (Cat# F-415XL; Thermo Scientific) on a Bio-Rad CFX384 Touch PCR system. Primer sequences are listed in Additional file 1 .
4
Chromatin Immunoprecipitation (ChIP) Protocol
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Cells were grown to 80 % confluence, washed twice in warm PBS, dissociated using Accutase (Cat# SCR005; Millipore), and resuspended in warm medium (DMEM F-12 Cat# 11320-033; Invitrogen) containing 0.1 volume crosslinking solution [89 (link)]. ChIP reactions were performed as described previously [35 (link)] followed by DNA purification using a Qiaquick PCR Cleanup kit (Cat# 28106; QIAGEN). Antibodies used include: anti-H3K4me3 (Cat# 04-745; Millipore), anti-H3K27me3 (Cat# 39155; Active Motif), anti-H3K9ac (Cat# 07-352; Millipore), and anti-H3K9me2 (Cat# 39239; Active Motif). Antibodies for modified histones were used at 1 µg/ChIP reaction. The concentration of IgG (Cat# SC-2027; Santa Cruz) was also used at 1 µg/ChIP. For analysis of candidate loci, real-time PCR was performed using the Dynamo Flash supermix (Cat# F-415XL; Thermo Scientific) according to the recommended protocol. Reactions were performed on a Bio-Rad CFX384 Touch PCR system. Primer sequences are listed in Additional file 3 .
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