GSM7731994: mLN Th KO1; Mus musculus; RNA-Seq
Small intestine was extracted from mice and Peyer's patches were removed. Then intestine was opened longitudinally, washed in PBS and cut into 0.5-1 cm pieces. The pieces were then incubated in ice cold PBS with 30 mM EDTA for 30 min and thoroughly washed in PBS. Afterwards they were cut into smaller pieces and digested three times for 10 min, 20 min and 30 min in RPMI 1640 containing 25 mM HEPES, 0,5 mg/ml Collagenase D and 10 µg/ml DNaseI. Disintegrated supernatants were collected in RPMI1640 + 10%FCS in between digestion steps. Cells were then filtered to remove clumps and Percoll density gradient centrifugation (40%/80%) was performed to purify lymphocytes. Spleen and lymph nodes were meshed through a 70 μm filter and then washed in DPBS. For spleens, lysis of erythrocytes was performed by incubating cells in 2 ml of ACK lysis buffer (0.15 M ammonium chloride, 10 mM potassium hydrogen carbonate, 1 mM EDTA-di sodium) for 2 min, before samples were washed and resuspended in DPBS. CD4 T cells were enriched prior to sorting using a CD4 T cell enrichment kit (Miltenyi) according to manufacturer's protocol. Cells were stained with antibodies against CD45, CD3 and CD4 and before sorting 7AAD was added to label dead cells. They were then sorted for CD45+CD3+CD4+7AAD- and depending on Foxp3 and RORgt reporter expression using the FACS Aria III. RNA isolation of bulk Treg populations was performed using the RNeasy Plus Micro Isolation Kit (Qiagen). Cells were sorted into lysis buffer containing β-mercaptoethanol and isolation was performed according to manufacturer's instructions. The SMART-Seq v4 ultra-low input RNA kit (Takara) was used for cDNA synthesis using the ERCC ExFold RNA Spike-In Mixes (Invitrogen). Library preparation was done using the Illumina Nextera XT DNA Library Preparation Kit. For bead cleanup AMPure XP (Beckman Coulter) were used.