NextSeq 500 paired end sequencing; CD1d-dependent immune suppression mediated by regulatory B cells through modulations of iNKT cells
CD1d-dependent immune suppression mediated by regulatory B cells through modulations of iNKT cells
C57BL/6 mice were from Harlan Laboratories (now Envigo Laboratories). μMT 69, Cd1d-/- 50, Il10-/- 70 mice were from The Jackson Laboratory. CD11c-DTR 71 mice were provided by C. Bennett, University College London, UK. Jα18-/- 49 mice were provided by A. Karadimitris, Imperial College London, UK. Il10ra-/- mice were provided by W. Muller, University of Manchester, UK.  IL-10 transcriptional reporter (Vert-X) mice were provided by C. Karp, Bill & Melinda Gates Foundation. All mice were on C57BL/6 background. Mice were age- and sex-matched, and used at 6-12 weeks of age. Splenic iNKT cells from B-Cd1d-/- and B-WT mice that received 2 micrograms of α-GalCer or vehicle as control were sorted by FACS. Sorted cells were >95% pure. Total RNA was isolated using PicoPure RNA Isolation Kit (Applied Biosystems) according to manufacturer's instructions. cDNA was synthesized and amplified using SMARTer v4 Ultra Low Input Kit (Clontech). 200pg of amplified cDNA was then converted to sequencing library using the Nextera XT Kit (Illumina, 12 cycles of PCR).  200pg of amplified cDNA was converted to sequencing library using the Nextera XT Kit (FC-131-1024, Illumina, 12 cycles of PCR)