title: GSM8757842: TCRa_PD1_KO3; Mus musculus; OTHER
library_construction_protocol: A total of 1 × 10⁵ donor-derived P14 cells were sorted and lysed in lysis buffer (100 mM Tris-HCl, pH 7.5; 500 mM LiCl; 10 mM EDTA, pH 8; 1.5% SDS) containing oligo-dT magnetic beads conjugated with Oligo-dT25. The mixture was incubated for five minutes to facilitate mRNA binding. Subsequently, the sample was placed on a magnetic rack to separate the beads, and the supernatant was discarded. The beads were washed twice with Wash Buffer A (10 mM Tris-HCl, pH 7.5; 0.15 M LiCl; 1 mM EDTA, pH 8; 0.1% SDS) and once with Wash Buffer B (10 mM Tris-HCl, pH 7.5; 0.15 M LiCl; 1 mM EDTA, pH 8) to remove non-specific contaminants. The supernatant was carefully removed after each wash. Elution of mRNA was achieved by adding 5 μL of Elution Buffer (10 mM Tris-HCl) and incubating the beads at 80°C for two minutes. The mixture was then subjected to magnetic separation, and the supernatant containing the extracted mRNA was collected. The RNA was reverse transcribed into cDNA using the Smart-Seq2 protocol,, followed by amplification for 13 cycles. A two-round nested PCR was performed with primers specific to TCRα and TCRβ.