GSM8581437: Guo-W1-18869; Mus musculus; RNA-Seq
The splenocytes were prepared freshly from CHD8-/- mice and their WT littermates. Natural regulatory T cells (nTregs) were isolated from spleens with a CD4+CD25+ Regulatory T Cell Isolation kit by AutoMACS Pro (Miltenyi Biotec, Auburn, CA). The purity of isolated Tregs was checked by FACS. Where indicated, splenocyte were stimulated with PMA (25 ng/ml) plus ionomycin (500 ng/ml) (Sigma, St Louis, MO) for 5 h with GolgiStop (BD Bioscience, San Jose, CA) in the last 2 h for intracellular cytokine staining. RNA seq: Splenic CD4+ T cells were isolated from WT and CHD8-/- mice by AutoMACS Pro. YFP+ cells were then sorted by FACS and analyzed for gene expression profiles by RNA-Seq. The Next Generation Sequencing technology (10X Genomics Next GEM 3v3.1 Assay) was performed by the Single Cell Genomics Core, Cincinnati Children's Hospital Medical Center. RNA-seq libraries were prepared using Illumina RNA-Seq Preparation Kit and sequenced using a HiSeq 2500 sequencer. Chip seq:Half million Tregs were used for ChIP-seq that was performed according to a previously described protocol with slight modifications (Boyer et al., 2005; Rada-Iglesias et al., 2011). In brief, Tregs were crosslinked with 1% formaldehyde at room temperature for 15 min with rotation, and then quenched with 125 mM glycine. Isolation of nuclei, extraction of chromatin and shearing with sonication were carried out as previously described (Cotney et al., 2013) using the Magna ChIP A/G kit (Millipore Sigma, Catalog#17-10085). Chromatin was immunoprecipitated by incubating soluble chromatin with 1.5 μg of CHD8 antibody (Bethyl Laboratories, # A301-224) or 1.5 μg of IgG antibody (Abcam, ab37355) at 4°C overnight. Magna ChIP® Protein A+G Magnetic Beads (Millipore Sigma, Catalog#16-663) were added next day and incubated for 2 hrs. CHD8-bound beads were washed in Low salt wash buffer, High salt wash buffer, LiCl wash buffer and TE buffer subsequentially. Harvested chromatin was then eluted from the beads, crosslinks were reversed and DNA was purified as previously described (Cotney et al., 2013), in which MinElute PCR Purification Kit (QIAGEN # 28204) was used for DNA purification. Libraries were prepared with the NEBNext® Ultra™ II DNA Library Prep Kit for Illumina (#E7645). Barcoded libraries were sequenced using a Novaseq 6000 sequencer at the DNA core of Cincinnati Children's Hospital Medical Center. ATAC seq:ATAC-seq was carried out as described by Buenrostro et al (Buenrostro et al., 2015). Briefly, ~50,000 sorted WT and CHD8-/- CD4+YFP+ Tregs were centrifuged at 500 x g for 20 minutes at 4 °C. Cells were lysed in ice cold lysis buffers (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% NP-40), centrifuged at 500xg at 40C for isolation of nuclei. Transposase reaction was performed by incubating the isolated nuclei with Tn5 transposase from the Nextera DNA Library Preparation Kit (Illumina) for 30 minutes at 37 °C. Transposed DNA was purified using QIAGEN mini elute DNA purification kit per manufacturer's instructions. PCR amplification and barcoding were done using forward (i5 Forward) and reverse (i7 Reverse) primers with specific index, NEB 2 x PCR Mix (New England Biolabs), and tagmented DNA. PCR conditions were as following: 72°C for 5 minutes, 98°C for 30 seconds, 5 cycles of 98°C for 10 seconds, 63°C for 30 seconds, and 72°C for 1 minute. After the first PCR, DNA size was selected with 0.5 x volume SPRI beads (Agencourt AMPure, Beckman Coulter), and cleaned up with 1.5 x SPRI beads. The second PCR (98°C for 30 seconds, followed by 5 cycles of 98°C for 10 seconds, 63°C for 30 seconds, and 72°C for 1 minute) was performed with same forward and reverse primers. Libraries were purified with 1.5 x SPRI beads, and concentration and ATAC DNA profiles were analyzed with the Bioanalyzer DNA High Sensitivity Kit (Agilent). Libraries were sequenced on Novaseq 6000 (Illumina), and with an average of 25 million paired-end reads per sample. RNA seq: The Next Generation Sequencing technology (10X Genomics Next GEM 3v3.1 Assay) was performed by the Single Cell Genomics Core, Cincinnati Children's Hospital Medical Center. RNA-seq libraries were prepared using Illumina RNA-Seq Preparation Kit and sequenced using a HiSeq 2500 sequencer. CHIP-seq:Barcoded libraries were sequenced using a Novaseq 6000 sequencer at the DNA core of Cincinnati Children's Hospital Medical Center. ATAC-seq: Libraries were sequenced on Novaseq 6000 (Illumina), and with an average of 25 million paired-end reads per sample.