title: GSM4478896: nTreg_ChIPseq_Input_sgBrd9+Foxp3; Mus musculus; ChIP-Seq
library_construction_protocol: For RNA-seq, total RNA was isolated using Quick-RNA Miniprep Kit (Zymo Research). For ChIP-Seq, 7.5e6 nTregs were collected and cross-linked first in 3mM disuccinimidyl glutarate (DSG) then 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 10-20 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 Foxp3, BRG1, BRD9, PHF10 and H3K27ac 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 and treated with Proteinase K and RNase A and the purified ChIP DNA was used for library generation for subsequent sequencing. For ATAC-seq, 50k nTregs per replicate were washed with cold PBS and resuspension buffer (RB; 10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2) before lysis in RB with 0.1% NP40. Cells were then incubated in transposition mix containing Tn5 transposase for thirty minutes at 37degC. ChIP-Seq libraries were generated using the NuGen Ovation Ultralow Library System V2 following the manufacturer's instructions. RNA-Seq libraries were prepared using Illumina TruSeq RNA Library Prep Kit v2 following the manufacturer's instructions. ATAC-seq -- purified DNA was ligated with adapters (NuGEN), amplified and size selected for sequencing.
attributes.tag: GEO Accession
attributes.value: GSM4478896