GSM3347716: Pol II ChIP-Seq in primary B cells – ZMYND8-F/F Cd19-Cre/+ rep 1; Mus musculus; ChIP-Seq
Sample information curated by ChIP-Atlas
Antigen
Antigen Class
RNA polymerase
Antigen
RNA polymerase II
Cell type
Cell type Class
Blood
Cell type
B cells
NA
NA
Attributes by original data submitter
Sample
source_name
Primary B cells
cell type
Splenocytes
genotype
Cd19-Cre/+ ZMYND8-F/F
antibody
anti-RNA Pol II polymerase (4H8, Abcam)
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
ChIP-Seq for ZMYND8 and RNA Pol II were performed as it follows. Splenocytes (72 h post-activation) or CH12 cells (48 h post-activation) were fixed with 1% (16% Formaldehyde Methanol-free, Thermo Scientific) at 37°C for 10 min followed by addition of 1/20 volume of 2.5 M glycine (dissolved in PBS pH 7.4) and swirling. Fixed cells were washed with cold PBS,centrifuged and aliquoted. 20 million cells were resuspended in 100 ul of 1% SDS of RIPA buffer supplemented with Complete EDTA free proteinase inhibitor (Roche) and sonication was performed with a Covaris S220 Focused Ultrasonicator at peak value 105, duty factor 5, cycle/burst 200 for 10 minutes. After sonication, samples were adjusted to 0.1% SDS final concentration by diluting with non-SDS containing RIPA buffer. Chromatin fragments were then pre-cleaned by incubation with Dynabeads Protein A (Invitrogen) with rotation at 4°C for 1 h, and immunoprecipitated with antibody-bound Dynabeads. 10 ug antibodies specific for ZMYND8 (Sigma-Aldrich) or RNA Pol II polymerase (4H8, Abcam) were used for each sample. DNA libraries were prepared by using Illumina compatible adaptors (Bio Scientific) and sequenced on an Illumina NextSeq 500 Sequencer.