title: RNA-seq of human iNKT cell lines primed with aGalCer vs Fusobacterium nucleatum
design: RNA-seq of human iNKT cell lines primed with aGalCer vs Fusobacterium nucleatum
library_construction_protocol: PB5 cells are derived in the following publication, supplementary Table S2 DOI: 10.1002/1878-0261.13104 from healthy adult donor peripheral blood. Monocyte derived dendritic cells (moDCs) were pulsed with alpha-Galactosylceramide (α-GalCer, CAS Number:158021-47-7)  (100ng/ml) or with heat-inactivated Fusobacterium nucleatum (ATCC25586) (4 × 10^5 CFU) and co-cultured with iNKT cells (2 × 10^5 cells) in a 2:1:4 iNKT:moDC:Fn ratio in RPMI-1640 supplemented with 10%FBS, Pen/Strep. After 24 h, iNKT cell activation status was estimated by intracellular staining and sorted to remove moDC before RNAseq experiments.  Healthy donors' peripheral blood buffy coats were obtained from San Matteo Hospital, Pavia, Italy, whereas intestinal specimens were obtained from IRCCS Policlinico Ospedale Maggiore, Milan, Italy. Institutional Review Board (Milan, Area B) approved the study with permission number 566_2015. All methodologies were under full compliance with the Declaration of Helsinki, and informed consent was obtained from all subjects. Peripheral blood mononuclear cells (PBMC) were isolated by density gradient centrifugation using Ficoll‐Paque as for standard protocol. Natural killer cells were isolated from PBMCs using the NK Isolation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany). Human lamina propria mononuclear cells (LPMCs) were isolated as previously described [53]. Briefly, the dissected intestinal mucosa was depleted of mucus and epithelial cells in sequential washes with Hanks' Balanced Salt Solution (HBSS, Euroclone, Pero, Italy) containing DTT (0.1 mmol·L−1, Sigma‐Aldrich, St Louis, MO, USA) and EDTA (1 mmol·L−1, Sigma‐Aldrich) and then digested with collagenase D (400 U·mL−1, Worthington Biochemical Corporation, Lakewood, NJ, USA) for 5 h at 37 °C. LPMCs were then separated with a Percoll gradient and cultured in RPMI‐1640 medium containing 5% human serum (Sigma‐Aldrich), penicillin/streptomycin, gentamicin, and 100 U·mL−1 IL‐2 (Proleukin). 1 × 106 iNKT cells were centrifuged and harvested in RLT buffer Total RNA (from 1 × 106 cells) was isolated with the RNeasy kit (Qiagen) and RNA quality was checked with the Agilent 2100 Bioanalyzer (Agilent Technologies). 0.5-1 μg were used to prepare libraries for RNA-seq with the Illumina TruSeq RNA Library Prep Kit v2 following the manufacturer's instructions.