RNA isolation and quantitative real time-PCR
To extract RNA from murine lung cells we used PeqGold RNA Pure according
to the manufacturer’s protocol (PeqLab, Erlangen, Germany). For reverse
transcription of RNA (1μg), we used the first strand cDNA synthesis kit
for RT-PCR (MBI Fermentas, Sat. Leon-Rot, Germany) followed by the
amplification by quantitative real-time PCR (qPCR) using SoFast EvaGreen
Supermix (Bio-Rad Laboratories, München, Germany). The qPCR itself was
performed in a CFX96 Touch Real-Time PCR Detection System (Bio-Rad
Laboratories) with a cycle of 2 min 98°C, 50 cycles at 5 s 95°C, 10 s
60°C, followed by 5 s 65°C and 5 s 95°. The primers and sequences used
for mouse were: mHPRT (5’- GCC CCA AAA TGG TTA AGG TT-‘3, 5’-TTG CGC TCA
TCT TAG GCT TT-‘3), mICOS (5’- GTG CAG CTT TCG TTG TGG TA –‘3, 5’ - TCA
GGG GAA CTA GTC CAT GC –‘3), mIL33 (5’- CCT CCC TGA GTA CAT ACA ATG
ACC- ‘3, 5’- GTA GTA GCA CCT GGT CTT GCT CTT –‘3), mFoxp3 (5’- AGA GCC
CTC ACA ACC AGC TA –‘3, 5’- CCA GAT GTT GTG GGT GAG TG –‘3), mIL10
(5’- CCA AGC CTT ATC GGA AAT GA –‘3, 5’- TTT TCA CAG GGG AGA AAT CG
–‘3), mRora (5’-TCT CCC TGC GCT CTC CGC AC-3’, 5’- TCC ACA GAT CTT GCA
TGG A –‘3), mST2 (5’-GCG GAG AAT GGA ACC AAC TA-‘3, 5’- AAG CAA GCT GAA
CAG GCA AT –‘3), mAREGB (5’- AGC TGA GGA CAA TGC AGG GTA-‘3, 5’-AGT GAC
AAC TGG GCATCT GG-‘3), mPD1 (5’-TCA AGG CATGGT CAT TGG TA-‘3, 5’-TAG GCC
ACA CTA GGG ACA GG-‘3).