Sample2
Mus musculus
Protocols: Cells were harvested by carefully removing media and adding 350 uL RLT lysis buffer supplemented with 1% v/v b-mercaptoethanol. Resting cells were pelleted by centrifugation and lysed in the same manner. Spleens were dissected post-mortem under sterile conditions. OT-I CTLs were differentiated and cultured from murine splenocytes in complete media, composed of RPMI 1640, 10 % heat inactivated FCS, 100 U/mL penicillin / 0.1 mg/mL streptomycin, 2 mM L-glutamine, 1 mM sodium pyruvate, 50 uM b-mercaptoethanol, and 20 ng/mL (≥ 100 U/mL) murine IL-2. Spleens were homogenized through a 70 μm filter and cultured in complete media with 10 nM SIINFEKL peptide for 3 days. Media was then changed and cells expanded daily until day 7. Flat-bottom tissue culture plates were coated with 1 μg/mL anti-CD3e (clone 145-2C11, BD Biosciences) in PBS at 37°C for 1 hour. Plates were then washed in PBS. Two million CTLs were stimulated on an antibody-coated 12-well plate for 10, 20, 40, and 60 minutes. For unstimulated control, cells were plated without antibody coating. Lysates were passed through a Qiashredder and frozen. RNA was extracted using the RNEasy Mini Kit and on-column DNA digestion with RNAse-free DNAse I. RNA quality was verified by Bioanalyzer 2100, and quantity was determined using a Qubit Fluorometer (v. 3). Libraries were prepared for sequencing with the TruSeq Stranded mRNA kit (Illumina).