GSM7140693: LTi-like ILC3 from the mLN of Bcl6-fl/fl control mice, biol rep 2; Mus musculus; RNA-Seq
For small intestine lymphocyte isolation, fat and Peyer's patches were removed and the small intestine was opened longitudinally and washed vigorously in PBS. Tissue was incubated for two concurrent rounds in PBS containing 1 mM EDTA, 1 mM dithiothreitol and 5 % FBS for 25 min at 37 °C with constant mixing. Then, tissues were washed in PBS and digested in complete RPMI with in 1 mg/ml collagenase VIII (Sigma-Aldrich) and 20 μg/ml DNase (Sigma-Aldrich) for 25 min at 37 °C. For mLN, fat was removed and lymph node was digested in 0.33mg/mL Liberase TL (Roche) and 1mg/ml DNase (Sigma-Aldrich)  for 25min at 37ºC on agitation. All the digested tissues were filtered through a 70 μm nylon strainer and stained for fluorescence-activated cell sorting. Live CD45+ B220- CD11c- CD11b- CD3- CD5- NK1.1- Ter119- TCRgd- TCRb- CD90.2+ CD127+ KLRG1- ILC were sorted as NCR+ ILC3 (NKp46+), or LTi-like ILC3 (CCR6+ ckit+)  into 250uL RL buffer using a BD FACS Aria II. Lysates were spun and frozen at -20ºC  overnight. Then, RNA was extracted using the Single Cell RNA Purification Kit (Norgen), including in-column DNA digestion, and frozen at -80ºC. RNA was amplified using SMART-Seq v4 Ultra Low Input RNA Kit for sequencing (Takara Bio USA, Inc., Mountain View, USA) following manufacturer's instructions. Libraries were prepared using NEBNext Ultra RNA Library Prep Kit (New England Biolabs, Ipswich, USA) following the manufacturer's recommendations.