RNA Pol II ChIP-seq Protocol

RNA polymerase II (Pol II) ChIP-seq was performed essentially with 5 biological replicates as previously described [36 (link)]. Fifteen micrograms of chromatin was immunoprecipitated overnight with 5 μg RNA Pol II antibody (Abcam, ab817) bound to protein A/G sepharose (Thermo Fisher, 10002D). Sequencing libraries were prepared by using NEBNext® Ultra™ II DNA Library Prep Kit for Illumina (NEB, E7645) and sequenced (75 nt single-end) on an Illumina NextSeq instrument.

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Shen J., Varshney D., Simeone A., Zhang X., Adhikari S., Tannahill D, & Balasubramanian S. (2021). Promoter G-quadruplex folding precedes transcription and is controlled by chromatin. Genome Biology, 22, 143.

Publication 2021

NextSeq instrument

Antibody Biological Chip seq Chromatin Dna library Protein a sepharose Rna polymerase ii

Corresponding Organization :

Other organizations : Cancer Research UK, University of Cambridge

Top 5 similar protocols

Variable analysis

independent variables

Chromatin immunoprecipitation (ChIP) with RNA polymerase II antibody

dependent variables

RNA polymerase II binding sites in the genome

control variables

Biological replicates (5)
Chromatin amount (15 micrograms)
RNA Pol II antibody concentration (5 μg)
Protein A/G sepharose for immunoprecipitation
NEBNext® Ultra™ II DNA Library Prep Kit for Illumina
Illumina NextSeq sequencing (75 nt single-end)

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

Annotations

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