GSM4154414: Input for H2BK120Ub ChIP-seq in Usp22KO mice Replicate 1; Mus musculus; ChIP-Seq
Sample information curated by ChIP-Atlas
Antigen
Antigen Class
Input control
Antigen
Input control
Cell type
Cell type Class
Blood
Cell type
Treg
NA
NA
Attributes by original data submitter
Sample
source_name
Primary Tregs from Usp22fl/fl/Foxp3-YFPCre
tissue
primary regulatory T cells (Tregs)
genotype/variation
Usp22 KO
antibody
none
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
For chromatin modifier ChIP-Seq, 4 e6 nTregs were collected and cross-linked first in 3mM disuccinimidyl glutarate (DSG) then in 1% formaldehyde. For histone modification ChIP-seq, 0.5-1 e6 nTregs were cross-linked in 1% formaldehyde. After quenching the excess formaldehyde with 125 mM glycine, the fixed cells were washed, pelleted and flash-frozen. Upon thawing, the cells were resuspended in lysis solution (50 mM HEPES-KOH pH 8, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP40, 0.25% Triton X-100 and incubated on ice for ten minutes. The isolated nuclei were washed with wash solution (10 mM Tris-HCl pH 8, 1 mM EDTA, 0.5 mM EGTA, 200 mM NaCl) and shearing buffer (0.1% SDS, 1 mM EDTA, 10 mM Tris-HCl pH 8) then sheared in a Covaris E229 sonicator for 10 minutes to generate DNA fragments between ~ 200-1000 bp. After clarification of insoluble material by centrifugation, the chromatin was immunoprecipitated overnight at 4C with antibodies against USP22, RNF20, H2AK119Ub, or H2BK120Ub then bound to Protein A+G Dynabeads (Invitrogen) in ChIP buffer (50 mM HEPES-KOH pH 7.5, 300 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% DOC, 0.1% SDS). Antibody bound DNA were washed, eluted, and treated with Proteinase K and RNase A and the purified ChIP DNA was used for library generation for subsequent sequencing. ChIP-Seq libraries were generated using the NuGen Ovation Ultralow Library System V2 following the manufacturer's instructions.