RNA-seq of Cdc42 knockout murine cytotoxic T lymphocytes
RNA-seq of Cdc42 knockout murine cytotoxic T lymphocytes
Cells were harvested by centrifugation into a cell pellet, and lysed with 350 μL RLT lysis buffer supplemented with 1% v/v beta-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 beta-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 nucleofected on day 5 after stimulation with CRISPR/Cas9 RNPs targeting Cdc42 or non-targeting control. Cells were harvested 72 hours after nucleofection. OT-I CTLs from six mice were nucleofected with CRISPR/Cas9 RNPs on day 5 post-stimulation. 72 h later, RNA was extracted from two million CTLs. 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 quality was verified by Bioanalyzer 2100 and quantity was determined using a Qubit Fluorometer (version 4). Libraries were prepared for sequencing with the TruSeq Stranded mRNA kit (Illumina).