RNA-seq of CD8+ OT-I T cells 7 dpi: UGCG-KO #3
Libraries were prepared by the VAI Genomics Core from 10 ng of total RNA using the SMART-Seq mRNA LP (with UMIs) low-input kit (Takara Bio USA) per the manufacturers protocol. Briefly, SMART-Seq mRNA oligo-(dT)s are used to isolate mRNA during the first stand synthesis, and SMART-Seq mRNA UMI TSOs uniquely label each transcript. After cDNA amplification, cDNA quality and quantity were determined via Agilent High Sensitivity DNA kit (Agilent Technologies). cDNA was then sheared via enzymatic fragmentation and dual indexes with sequencing adapters were added via PCR amplification using a Unique Dual Index Kit (Takara Bio USA). The quality and quantity of the libraries were determined using a combination of Agilent High Sensitivity DNA kit, QuantiFluor dsDNA System (Promega), and Kapa Illumina Library Quantification qPCR assays (Kapa Biosystems).  Individually indexed libraries were pooled and 75 bp, paired-end sequencing was performed on an Element AVITI sequencer using a mid-output, 150 bp sequencing kit (Element Biosciences) to an average depth of 70M reads per sample. Base calling was performed on instrument and AVITI OS (v2.6.2) output was demultiplexed and converted to FASTQ format with Element Biosciences Bases2fastq (v1.7.0).