RNA-seq of murine cytotoxic T lymphocytes in response to myosin IIA inhibition using para-nitroblebbistatin
RNA-seq of murine cytotoxic T lymphocytes in response to myosin IIA inhibition using para-nitroblebbistatin
Cells were harvested by centrifugation into a cell pellet, and lysed with 350 μL RLT lysis buffer supplemented with 1% v/v β-mercaptoethanol. Spleens were dissected post-mortem under sterile conditions. Effector OT-I CTLs were differentiated and cultured from murine splenocytes as follows. Naïve OT-I splenocytes were stimulated with 10 nM ovalbumin peptide, SIINFEKL (OVA) for 3 days in complete mouse T cell media: RPMI 1640 medium with 10% FBS, 50 μM β-mercaptoethanol, 10 U/ml recombinant murine IL-2, 2 mM L-Glutamine, 1 mM sodium pyruvate, 100 U/ml penicillin and 100 μg/ml streptomycin. Cells were pelleted and resuspended in fresh media from 3 days after stimulation, expanded in CTL media and used at day 7 after stimulation. 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 OT-I CTLs were stimulated (or not stimulated) for 1 hour on anti-CD3e coated plate, treated with either 20 μM pNB or DMSO. 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, according to the manufacturer's protocol with on-column DNA digestion with RNase-free DNase I. RNA samples from 6 biological replicates from the same litter (3 males and 3 females, aged between 11 to 14 weeks) were stimulated in 4 independent experiments. RNA extraction from all 24 cell pellets were performed on the same day. RNA quality was verified by Tapestation 4150 and quantity was determined using a Qubit Fluorometer (version 4). Libraries were prepared for sequencing with the TruSeq Stranded mRNA kit (Illumina).