Illumina HiSeq 2500 paired end sequencing; Fibroblastic reticular cells serve as primary immune checkpoint in visceral adipose tissues and secure peritoneal immunity
Fibroblastic reticular cells serve as primary immune checkpoint in visceral adipose tissues and secure peritoneal immunity
Ccl19-Cre mice were crossed with R26-stop-EYFP (Ccl19-EYFP). To specifically delete Myd88 from fibroblastic stromal cells (FRCs), Ccl19-EYFP mice were crossed with Myd88fl/fl mice (Ccl19-EYFP Myd88fl/fl ). Ccl19-EYFP and Ccl19-EYFP Myd88fl/fl mice were immunized or not with 10µg of Salmonella typhi OmpC/F porin intraperitoneally. Omentum was collected from naive or immunized mice at day 4 post-administration. CD45- CD31- Ter119- EYFP+ fibroblastic reticular cells from Ccl19-EYFP and Ccl19-EYFP Myd88fl/fl were sorted from independent mice with a Bio-Rad S3 cell sorter and were collected in 200 µl Eppendorf tubes containing 100 µl RNAlater reagent (Qiagen) to preserve the RNA after sorting.  Note: BAC-transgenic C57BL/6N-Tg(Ccl19-cre)489Biat mice (Ccl19-Cre mice) Ccl19-Cre were crossed with  Gt(ROSA)26Sortm1(EYFP)Cos (Ccl19-EYFP) Ccl19-EYFP were crossed with the Myd88tm1Defr (Ccl19-EYFP Myd88fl/fl) RNA extraction was performed with Quick-RNA Micro-Prep (Zymo Research). Reverse transcription and cDNA library generation was done using the Ovation SoLo RNA-Seq System (NuGEN) as described by the manufacturer. cDNA libraries were quantified by KAPA library Quantification kit (KAPA Biosystems) and Agilent Tape station in the Functional Genomics Center Zurich, Zurich, Switzerland.