Qiashredder and rneasy kit

Total cellular RNA was isolated using the QIA Shredder and RNeasy kit (Qiagen, Valencia, CA), as described by the manufacturer’s protocol. Reverse transcription was performed using Applied Biosystems high-capacity cDNA reverse transcription kits (Invitrogen, Carlsbad, CA) with random primers. To quantify gene expression, quantitative real-time PCR was performed in the Bio-Rad IQ5 system (Bio-Rad Laboratories, Hercules, CA) using Finnzymes SYBR green I dye (New England Biolabs, Ipswich, MA), and sequence-specific primers, as indicated in Table 1. The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used as an internal control. The reactions were performed under the following conditions: 95 °C for 3 min, followed by 45 cycles at 94 °C for 20 seconds, 60 °C for 30 seconds, and 72 °C for 20 seconds. The mRNA level of each gene was normalized to GAPDH levels to obtain mRNA arbitrary units (fold change).

GE of all prostate cancer and normal prostate cell lines at baseline (no treatment) was assessed by RNA-seq. Furthermore, the effects of METRO-TOPO and CONV-TOPO exposure for 6 weeks on AR^Low mCRPC/NEPC PC-3 tumor model (3D spheroid) were assessed using RNA-seq. Pre- and post-drug exposure, as described above, tumor cells were harvested, and high-quality RNA was extracted using QIAshredder and RNeasy kit (Qiagen). RNA concentration and integrity were assessed using a Nanodrop-8000 spectrophotometer and Agilent 2100 Bioanalyzer. An RNA integrity number threshold >8 was applied, and RNA-seq libraries were constructed using Illumina TruSeq RNA Sample Preparation kit v2. Libraries were then size-selected to generate inserts of approximately 200 bp. RNA-seq was performed on Illumina's NovaSeq platform using a 150 bp paired-end protocol with a depth of >20 million reads per sample. Average quality scores were above Q30 for all libraries in both R1 and R2 (29 (link)). AA cell line RNA was isolated from cultured cells using TRIzol Reagent (Sigma Life Sciences) following the manufacturer's protocol (27 (link)). Library preparation, quality control, and sequencing of extracted RNA were performed by the Center for Pharmacogenomics and Single-Cell Omics (AUPharmGX).

Total RNA was extracted from thymocytes and selected CD4⁺ T cells using a QIAshredder and RNeasy kit (Qiagen) followed by reverse transcription using the SuperScript III (Invitrogen) kit. Quantitative PCR was performed using the TaqMan Gene Expression assays (Applied Biosystems) on a StepOnePlus system (Applied Biosystems). TaqMan gene expression probes were used for gene analysis of mouse Shcbp1, ShcA, HPRT, Shcbp1-L, Fbox10, Fbox11 and IL-2. Each sample was performed in duplicate, target transcripts were normalized to HPRT mRNA as an internal control gene, and the relative expression of each target gene was calculated using the comparative cycling method with StepOne v2.1 software (Applied Biosystems).