Illumina HiSeq 2000 paired end sequencing; Foxp3 regulates gene expression mostly via its proline-rich interaction region independent of its own DNA-binding domain
Foxp3 regulates gene expression mostly via its proline-rich interaction region independent of its own DNA-binding domain
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BALB/c CD4+CD25- T cells were isolated by negatively depleting splenocytes stained with FITC-conjugated depletion cocktail antibodies including anti-CD8, anti-CD19, anti-CD11b, anti-CD11c, anti-Ly6G, anti-TER119 and anti-CD25 using autoMACS machine (Miltenyi Biotc). Isolated mouse primary T cells were cultured in RPMI-1640 medium supplemented with 10% FCS, antibiotics and 50uM 2-mercaptoethanol. Primary T cells were transduced with virus carrying Foxp3 and its mutants. Transduced cells were allowed to rest for 48 h in RPMI medium with IL2. The resting cell expressing transduction marker rCD8a were sorted by FACS or autoMACS. For re-activation sample, sorted cells were activated with plate-coated anti-CD3 (5 ug/ml), soluble anti-CD28 (1 ug/ml) for 8 h. mRNA isolation was performed using uMACS mRNA isolation kit (Miltenyi Biotec). 5x106 cells of each sample were lysed for mRNA isolation. cDNA synthesis was performed using the uMACS cDNA synthesis module (Miltenyi Biotec). Second strand was synthesized using E.coli DNA polymerase I (NEB), E.coli DNA ligase (NEB) and E.coli RNaseH (NEB) in second strand synthesis buffer (Invitrogen) at 16C for 2.5 h. Double strand DNA was fragmented by sonication using Bioruptor UCD-400 (Diagenode), with 20 cycles of (30 sec On; 30 second off). Fragmented samples were end-repaired using Klenow large fragment (NEB), T4 DNA polymerase(NEB) and T4 polynucleotide kinase (NEB) at 20C for 30 min. Purified blunt-ended DNA was 3' adenylated using 3' greater than 5' exo- Klenow fragment (NEB) at 37C for 30 min. After purification, adapters containing 6 bp barcodes from sample prep oligonucleotide kit (Illumina) were ligated to 3' A-tailed DNA fragment at 20C for 30 min using T4 DNA ligase (NEB). Each DNA sample was amplified via 18-cycle PCR using universal primers from the sample prep oligonucleotide kit. The amplified library was size-selected from 200-400 bps using 2% agarose gel and quantified with a high-sensitivity DNA chip (Agilent) using agilent bioanalyzer.