RNA-seq of mouse CD8+ T cells treated with 25-HC versus DMSO control
RNA-seq of mouse CD8+ T cells treated with 25-HC versus DMSO control
The cells were harvested, stained at 4°C for 30 minutes for surface markers, and subsequently  sorted on a BD FACSAria IIu. Eight-week-old male C57BL/6J mice were euthanized by CO₂ asphyxiation. The abdominal cavity was opened by a midline incision. The spleen was identified, gently dissected free from surrounding connective tissue at the hilum. Naïve CD8+ T cells were purified from the spleens of C57BL/6 mice by negative selection. The cells were maintained in complete RPMI-1640 medium, supplemented with 10% FBS, 2 mM L-glutamine, 1% penicillin-streptomycin, 10 mM HEPES, and 50 μM 2-mercaptoethanol. End repair, A-tailing, adapter ligation, and PCR amplification using Illumina-compatible reagents. Total RNA was extracted from the tissue sample using Trizol according to the user manual. 5' RACE was performed for reverse transcription. The reverse transcription products were amplified by PCR with corresponding specific primers. PCR products were purified by DNA magnetic beads. PCR purified product is terminal repair (including 5 'terminal phosphorylation and 3' terminal plus' A ') by the End Prep Enzyme Mix, with sequencing adapters at both ends. Then DNA Clean Beads were purified and amplified with P5 and P7 primers. The PCR products were cleaned up using beads, validated using an Qsep100 (Bioptic, Taiwan, China), and quantified by Qubit3.0 Fluorometer  (Invitrogen, Carlsbad, CA, USA). Barcoded sequencing libraries were constructed using the TruSeq Stranded mRNA Kit (Illumina).