GSM1666196: Foxp3 Il10 cells; Mus musculus; RNA-Seq
After removal of the Peyer’s patches, we isolated intraepithelial lymphocytes (IELs) and lamina propria lymphocytes (LPLs) by incubation with 1mM DTT at 37 °C for 30 min (for IEL), followed by further digestion with collagenase IV and DNase at 37 °C for 1 h (for LPL). We then further separated cells with a Percoll gradient. Unless otherwise indicated, we isolated cells from the small intestine (duodenum, ileum and jejunum) of mice treated with antibodies to CD3. We isolated intestinal lymphocytes from two independent experiments, each using 5 mice injected with anti-CD3 mAb. Th17, exTh17, Tr1 exTh17, Tr1, Foxp3 Treg and Foxp3 IL-10+ Treg cell populations were FACS-sorted from these two independent experiments and the cells of each population were pooled before the analysis. Around 5,000 cells for each population were processed. After sorting, the cells were washed 2 times (1,500rpm, 2min, 4°C) with 1ml phosphate-buffered saline (PBS) and finally suspended in 2.5µl PBS (contain 0.5µl RNaseOut (Invitrogen) and 0.5µl dithiothreitol (DDT) (Invitrogen)). After that, the cytoplasm RNA was isolated as described previously (Guo, S. et al. Cell 156 (4), 649 (2014); Pan, X. et al., Proc Natl Acad Sci U S A 110, 594 (2013).) Briefly, 2.5µl of 2x selected cytoplasm lysis buffer (SCLB) was added and the cells were lysed by pipetting up and down for 5 times. The entire lysate solution was spun at 8000rpm for 5 min in a chilled centrifuge (4°C) and the supernatant (~5µl), which contained the total cytoplasm RNA, was transferred to a PCR tube strip with individually attached dome caps (USA Scientific). The mRNA selection, reverse transcription and cDNA amplification was performed as described previously with some modifications (Guo, S. et al. Cell 156 (4), 649 (2014); Pan, X. et al., Proc Natl Acad Sci U S A 110, 594 (2013). Briefly, 5’-phosphorylated oligo-GdT24 (pGdT24) primer was used to selectively reverse transcribe mRNAs. The first strand cDNA was synthesized with Superscript Reverse Transcriptase III (invitrogen, 10u/µl). Then, the double-stranded cDNA (dscDNA) was generated. The cDNA was purified with the Genomic DNA Clean & Concentrator kit (Zymo). Afterwards, several steps including DNA end-blunting, 5’-end phosphorylation and ligation were performed with The End-It DNA End-Repair Kit (Epicentre) with T4 DNA ligase (Epicentre). The product was directly amplified using REPLI-g UltraFast Mini Kit (QIAGEN) and purified using the same Genomic DNA Clean & Concentrator kit (Zymo) column. Finally, 3 to 5 µg (up to 8 µg) of amplified cDNA derived from mature mRNA was obtained, which was then evaluated by PCR and fragmented to construct sequencing library. We follow the standard Illumina HiSeq2000 protocol to make the library. Briefly, the amplicon was fragmented to an approximately 200-500 bp size range by a Bioruptor Sonicator (Diagenode). After purification with DNA Clean & Concentrator kit (Zymo), end-repairing, 3’-A tailing and ligation with adaptor was performed. Then, a 50bp range DNA (250-300bp) was selected by gel electrophoresis (E-gel EX 2%, invitrogen) and barcode added by PCR using Phusion High-Fidelity DNA polymerase (NEB) for 8 cycles. The product was size selected again and the DNA concentration was quantitated by a Bioanalyzer (Agilent). Multiple samples were mixed and loaded to the Hi-Seq2000 for sequencing performed with 50bp single end reads.
GEO Accession
GSM1666196