Anti-BRD4 and anti-H3K27ac Chip-qPCR of POU5F1B superenhancer was performed, respectively. The forward and reverse primers are listed in supplementary table 2 . Chromatin immunoprecipitation (ChIP) assay was performed according to the protocol developed by Upstate Biotechnology. Briefly, chromatin lysates were prepared from hepatocytes after crosslinking with 1% formaldehyde. The samples were precleared with Protein-G agarose beads and immunoprecipitated using anti-H3K27ac antibody (Abcam, #cat: ab4729), anti-BRD4 antibody (Abcam, #cat: ab128874), or control anti-IgG (Abcam, #cat: ab172730) in the presence of BSA. Beads were extensively washed before reverse crosslinking. DNA was purified using a PCR Purification Kit and subsequently analyzed by qPCR.
Control anti igg
Manufactured by Abcam
Control anti-IgG is a control reagent used to validate the performance of assays that detect or measure immunoglobulin G (IgG) in a sample. It provides a consistent and reliable control to ensure the accuracy and reproducibility of IgG-based assays.
Automatically generated - may contain errors
Lab products found in correlation
Anti brd4 antibody, by Abcam (1 mentions)
Anti h3k27ac antibody, by Abcam (1 mentions)
Rabbit anti ha antibody, by Abcam (1 mentions)
Anti atf4, by Abcam (1 mentions)
Dynabeads protein g immunoprecipitation kit, by Thermo Fisher Scientific (1 mentions)
Anti argonaute2 ago2 antibody, by Abcam (1 mentions)
4 protocols using control anti igg
1
POU5F1B Superenhancer Binding Analysis
2
Chromatin Immunoprecipitation of Ngn3 Variants
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ChIP experiments were performed on mPAC cell extracts, 24 hours after transfection with either HA-tagged WT or 6S-A Ngn3. Chromatin-protein complexes were cross-linked with 1% formaldehyde. Five micrograms of rabbit anti-HA antibody (Abcam) or control anti-IgG (Abcam) were used per ChIP reaction and quantified using SYBR Green mix. The signal over background normalisation method was used to quantify immunoprecipitated DNA. Primer list is provided in Table S2 .
3
Chromatin immunoprecipitation of ATF4
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ChIP assay was implemented using EpiQuik Chromatin immunoprecipitation Assay Kit (AmyJet, Wuhan, China) following the manufacturer’s protocols. Briefly, HASMCs were fixed with 1% formaldehyde and then treated with glycine for 5 mins. Afterwards, the chromatin was sonicated into DNA fragments (200–400 bp), and anti-ATF4 and control anti-IgG (Abcam) were used to precipitate the chromatin. Then quantification of the precipitated DNA was performed using RT-qPCR.
4
RNA Immunoprecipitation of Ago2 Complexes
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With the manufacturer’s instructions, RNA Immunoprecipitation experiment was conducted using Dynabeads Protein G Immunoprecipitation Kit (Thermo). In brief, transfected DU145 and LNCap cells were lysed with RIP lysis buffer. And then we used magnetic beads conjugated with anti-Argonaute 2 (Ago2) antibody (Abcam) or control anti-IgG (Abcam) to enrich RNA-protein complexes. In addition, RNA fragments isolated from the mixture with TRIzol Reagent were detected by qRT-PCR.
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