Nanodrop fluorimeter

Total RNA was extracted from peripheral-blood leukocytes using the TRIzol™ Plus RNA Purification Kit (ThermoFisher Scientific, Waltham, Massachusetts, USA), and all steps followed the protocol recommended by the manufacturer. The concentration of extracted RNA was determined using a NanoDrop™ fluorimeter (ThermoFisher Scientific, Waltham, Massachusetts, USA) according to the manufacturer's instructions. All total RNA samples were diluted to 50 ng/µL for complementary DNA (cDNA) synthesis.

RNAs were extracted using TRIzol Reagent (Invitrogen) and subsequently purified using silica membrane spin columns from Nucleospin RNA (Mechery Nagel). RNA quantity and purity were assessed using a NanoDrop fluorimeter (Thermo-Fisher). 4 µg of total RNA were reverse-transcribed using the random hexamers-based High Capacity cDNA Reverse-Transcription Kit (Applied Biosystem), according to the manufacturer’s instructions.
Gene expression of p53 targets was measured using qPCR. The reaction was performed in a final volume of 20 µl, using the LightCycler 480 SYBR Green I Master mix (Roche, Monza, IT), 0.5 µl of diluted cDNA (1:100), and 0.5 nM of each primer.
Normalization of cDNA loading was obtained by running all samples in parallel using human GAPDH as a loading control. The cDNA was amplified using an initial denaturation and activation step at 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s, 60 °C for 1 min, and 95 °C for 15 s. The sequences of the primers used are provided in Supplementary Table 1.

TCR sequencing was carried out by Adaptive Biotechnologies according to standard company protocols. Experimentally, mice from each group were sacrificed and blood draw was obtained through cardiac puncture. Isolation of genomic DNA was performed in the laboratory using a Quiagen DNEasy Blood and Tissue Kit and gDNA quality and concentration was confirmed using a Nano‐Drop fluorimeter (Thermo). Isolated gDNA was then sent to Adaptive headquarters where proprietary primer sets were utilized to isolate and amplify the TCRB gene locus within in mouse genome. Sequencing of the resulting cDNA libraries was performed at Adaptive's facility and PCR duplication bias was eliminated using established company quality controls. Both raw and quantified data were transferred back to the laboratory using Adaptive's data analysis cloud. Raw data were analyzed using MiXCR (Bolotin et al., 2015 (link)) while quantified data were mined and plotting using R (version 4.1).