alias: GSM4154424
title: GSM4154424: Input for histone ChIP-seq in sgUsp22 Rep1; Mus musculus; ChIP-Seq
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.
identifiers.namespace: GEO
identifiers.id: GSM4154424
attributes.tag: GEO Accession
attributes.value: GSM4154424