ChM_Input_A
Mus musculus
Protocols: 20 million cells from each sample were crosslinked in 1% HCHO (prepared in 1X DPBS) at room temperature for 10 minutes, and HCHO was quenched by the addition of glycine at a final concentration of 0.125 M. Cells were pelleted at 4°C at 2000 x g, washed with ice-cold 1X DPBS twice, and snapped frozen in liquid nitrogen. The cell pellets were stored in -80°C until the experiments were performed. ChIPmentation was performed according to the version 1.0 of the published protocol (Schmidl et al., 2015b) with some modifications at the ChIP stage. Briefly, cell pellets were thawed on ice, and lysed in 300 l ChIP Lysis Buffer I (50 mM HEPES.KOH, pH 7.5, 140 mM NaCl, 1 mM EDTA, pH 8.0, 10% Glycerol, 0.5% NP-40, 0.25% Triton X-100) on ice for 10 minutes. Then cells were pelleted at 4°C at 2000 x g for 5 minutes, and washed by 300 l ChIP Lysis Buffer II (10 mM Tris.Cl, pH 8.0, 200 mM NaCl, 1 mM EDTA, pH 8.0, 0.5 mM EGTA, pH 8.0), and pelleted again at 4°C at 2000 x g for 5 minutes. Nuclei were resuspended in 300 l ChIP Lysis Buffer III (10 mM Tris.Cl, pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% Sodium Deoxycholate, 0.5% N-Lauryolsarcosine). Chromatin was sonicated using Bioruptor Pico (Diagenode) with 30 seconds ON/30 seconds OFF for 5 cycles. 30 l 10% Triton X-100 were added into each sonicated chromatin, and insoluble chromatin was pelleted at 16,100 x g at 4°C for 10 minutes. 1 l supernatant was taken as input control. The rest of the supernatant was incubated with 10 l Protein A Dynabeads (Invitrogen) pre-bound with 1 g XBP1 antibody (XBP1 (M-186)X - Santa cruz), in a rotating platform in a cold room overnight. Each immunoprecipitation (IP) was washed with 500 l RIPA Buffer (50 mM HEPES.KOH, pH 7.5, 500 mM LiCl, 1 mM EDTA, 1% NP-40, 0.7% Sodium Deoxycholate, check components) for 3 times. Then, each IP was washed with 500 l 10 mM Tris, pH 8.0 twice, and resuspended in 30 l tagmentation reaction mix (10 mM Tris.Cl, pH 8.0, 5 mM Mg2Cl, 1 l TDE1 (Nextera)). Then, the tagmentation reaction was put on a thermomixer at 37°C for 10 minutes at 800 rpm shaking. After the tagmentation reaction, each IP was washed sequentially with 500 l RIPA Buffer twice, and 1X TE NaCl (10 mM Tris.Cl, pH 8.0, 1 mM EDTA, pH 8.0, 50 mM NaCl) once. Elution and reverse-crosslinking was done by resuspending the beads with 100 l ChIP Elution Buffer (50 mM Tris.Cl, pH 8.0, 10 mM EDTA, pH 8.0, 1% SDS) on a thermomixer at 65°C overnight, 1,400 rpm. DNA was purified by MinElute PCR Purification Kit (QIAGEN, cat no. 28004 and eluted in 12.5 l Buffer EB (QIAGEN kit, cat no 28004), which yielded ~10 l ChIPed DNA. ChIPed DNA before amplification was purified using Qiagen MinElute PCR purification kit. After library amplification, DNA was purified using AmpureXP beads with a 0.5X upper cutoff and 1.2X lower cutoff. The library preparation reactions contained the following: 10 l purified DNA (from above), 2.5 l PCR Primer Cocktails (Nextera DNA Library Preparation Kit, Illumina Cat no. FC-121-1030), 2.5 l N5xx (Nextera Index Kit , Illumina cat no.FC-121-1012), 2.5 l N7xx (Nextera index kit , Illumina cat no. FC-121-1012), 7.5 l NPM PCR Master Mix (Nextera DNA Library Preparation Kit, Illumina Cat no. FC-121-1030) PCR was set up as follows: 72°C, 5 mins; 98°C, 2 mins; [98°C, 10 secs, 63°C, 30 secs, 72°C, 20 secs] x 12; 10°C hold
ENA first public
2018-06-30
ENA last update
2017-12-20
External Id
SAMEA104445854
INSDC center alias
Wellcome Trust Sanger Institute
INSDC center name
Wellcome Trust Sanger Institute
INSDC first public
2018-06-30T17:01:49Z
INSDC last update
2017-12-20T10:50:00Z
INSDC status
public
Submitter Id
E-MTAB-6327:ChM_Input_A
broker name
ArrayExpress
cell type
CD4-positive helper T cell
common name
house mouse
sample name
E-MTAB-6327:ChM_Input_A
stimulus
interleukin-4 (10 nanogram per milliter for 5 days)