GSM2219814: muscle Pol II WT; Mus musculus; ChIP-Seq
Sample information curated by ChIP-Atlas
Antigen
Antigen Class
RNA polymerase
Antigen
RNA polymerase II
Cell type
Cell type Class
Muscle
Cell type
Skeletal muscle
NA
NA
Attributes by original data submitter
Sample
source_name
ChIP DNA
strain background
C57/B6
genotype/variation
wild type
tissue
Hind-limb muscles
chip antibody
Pol II
chip antibody vendor
Santa Cruz
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Hind-limb muscles from wild-type and mutant (HSA::Cre-ERT2::Tead4lox/lox , Conditional muscle-specific Tead4 knockout) mice were minced and homogenised, fixed in 1% formaldehyde for 10 min at room temperature. Cross-linking was stopped by the adding Glycine to final concentration 0.125 M. The tissue lysate was further homogenized in hypotonic buffer in order to obtain nuclei, the nuclear pellet was washed once in hypotonic buffer and then resuspended in sonication buffer. Nuclear lysates were sonicated using Covaris E220 to obtain DNA fragments <500 bp. Chromatin was pretreated with blocked protein G sepharose beads. Subsequently, samples were incubated overnight at 4°C with antibodies to AcH3K27 (Active Motif) or RNA PoI II antibody (Santa cruz). Blocked beads were then added and the mixture was incubated for 2 h at 4°C. Prot G beads were washed, protein-DNA complexes were eluted from the beads and decrosslinked, and DNA was recovered by phenol chloroform extraction and ethanol precipitation after treatment with ribonuclease A (Abcam) and proteinase K. ChIP-seq libraries were prepared using NEXTflex ChIP-Seq Kit (#5143-02, Bioo Scientific) following the manufacturer's protocol (V12.10) with some modifications. Briefly, 10 ng of ChIP enriched DNA or INPUT DNA were end repaired using T4 DNA polymerase, Klenow DNA polymerase and T4 PNK, then size selected and cleaned-up using Agencourt AMPure XP beads (#A63881, Beckman). A single ‘A’ nucleotide was added to the 3’ ends of the blunt DNA fragments with a Klenow fragment (3' to 5'exo minus). The ends of the DNA fragments were ligated to double stranded barcoded DNA adapters (NEXTflex ChIP-Seq Barcodes - 6, #514120, Bioo Scientific) using T4 DNA Ligase. The ligated products were enriched by PCR (2 min at 98°C; [30 sec at 98°C, 30 sec at 65°C, 60 sec at 72°C] x 14 cycles; 4 min at 72°C) and cleaned-up using Agencourt AMPure XP beads.