GATA3-24h-B
Mus musculus
Protocols: CD4+CD62L+ naive T cells were purified from spleens of wild-type C57BL/6JAX adult (6 - 8 weeks) mice using the CD4+CD62L+ T Cell Isolation Kit II (Miltenyii #130-093-227). Cell culture plates were coated with anti-CD3e antibody (1 ug/ml, eBioscences #16-0031-81) in 1X DPBS (Gibco) at 4°C overnight. Purified naive T cells were seeded at a concentration of 1 M cells/ml on the coated plates in IMDM (Gibco) with 10% heat-inactivated FBS (Sigma), supplied with 5 ug/ml anti-CD28 (eBioscience #16-0281-86) with (Th2) or without (Th0) 10 ng/ml mouse recombinant IL-4 (R&D Systems #404-ML-050). Cells were cultured in plates for up to 72 hours. Total RNA was purified by Qiagen RNeasy Mini Kit according to manufacturer's instruction, and concentration was determined by a Nanodrop. A total of 500 ng RNA was used to prepare sequencing libraries using KAPA Stranded mRNA-seq Kit (KAPA #07962193001) according to manufacturer's instructions. Sequencing was performed on an Illumina HiSeq 2000 (125bp PE, v4 chemistry). ChIPmentation was used to investigate the TF binding sites. 1 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 (http://www.medical-epigenomics.org/papers/schmidl2015/) with some modifications at the ChIP stage.Briefly, cell pellets were thawed on ice, and lysed in 300 ul 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 ul 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 ul 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-Lauroylsarcosine). Chromatin was sonicated using Bioruptor Pico (Diagenode) with 30 seconds ON/30 seconds OFF for 10 cycles. 30 ul 10% Triton X-100 was added into each sonicated chromatin, and insoluble chromatin was pelleted at 16,100 x g at 4°C for 10 minutes. 1 ul supernatant was taken as input control. The rest of the supernatant was incubated with 10 ul Protein A Dynabeads (Invitrogen) pre-bound with 1 ug anti-FLAG in a rotating platform in a cold room overnight. Each immunoprecipitation (IP) was washed with 500 ul 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 ul 10 mM Tris, pH 8.0 twice, and resuspended in 30 ul tagmentation reaction mix (10 mM Tris.Cl, pH 8.0, 5 mM Mg2Cl, 1 ul 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 ul 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 were done by resuspending the beads with 100 ul 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, #28004 and eluted in 12.5 ul Buffer EB (QIAGEN kit, #28004), which yielded ~10 ul ChIPed DNA. The library preparation reactions contained the following: 10 ul purified DNA (from above) 2.5 ul PCR Primer Cocktails (Nextera DNA Library Preparation Kit, Illumina #FC-121-1030) 2.5 ul N5xx (Nextera Index Kit , Illumina #FC-121-1012) 2.5 ul N7xx (Nextera index kit , Illumina #FC-121-1012) 7.5 ul NPM PCR Master Mix (Nextera DNA Library Preparation Kit, Illumina #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 holdThe amplified libraries were purified by double AmpureXP beads purification: first with 0.5X bead ratio, keep supernatant, second with 1.4X bead ratio, keep bound DNA. Elution was done in 20 ul Buffer EB (QIAGEN).1 ul of library was run on an Agilent Bioanalyzer to see the size distribution