Oligotex kit

The isolated PBMCs were washed three times with ice-cold PBS then 1 ml TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) was added to extract the total RNA according to the manufacturer's instructions. An Oligotex kit (Qiagen, Inc., Valencia, CA, USA) was used to purify the mRNA from total RNA according to the manufacturer's protocol. The purified mRNA was converted to cDNA using SuperScript III Reverse Transcriptase, DNase, buffers and dNTPs (all from Thermal Fisher Scientific, Inc.). A degenerated V region primer (5′-GCIITKTIYTGGTAYMGACA-3′), which was designed to cover the majority of the TRBV genes, and a reverse primer (5′-GCACCTCCTTCCCATTCAC-3′) covering both TRBC genes was used as previously reported (18 (link),19 (link)). Each 50 µl RT-qPCR reaction contained 2 µl cDNA, 12 µl ddH₂O, 25 µl Premix Ex Taq (Takara Bio, Inc., Otsu, Japan), 1 µl forward primer, 1 µl reverse primer. The RT-qPCR cycling conditions were used as follows: 94°C for 10 min; 40 cycles of 94°C for 30 sec, 50°C for 30 sec, and 72°C for 30 sec; and a final 10 min extension at 72°C and 4°C hold for 30 sec. The PCR reaction was performed using an ABI 7500 Real-Time PCR System (Thermo Fisher Scientific, Inc.).

Total RNA was extracted from growing MCF7 cells as described and enriched for mRNA using Oligotex kit (QIAGEN). Equal volumes of reconstituted RNA were separated on 1.2% agarose gel supplemented with 0.66% (vol/vol) formaldehyde and transferred onto Amersham Hybond-N⁺ membrane (GE Healthcare). RNA probes targeting Renilla or Firefly mRNAs were produced by in vitro transcription (T7 high yield kit, NEB) of PCR fragments encoding for sequences complementary to these transcripts (see Table S4 for used oligos) using ³²P(α)-UTP. Following cleaning of the probes using MicroSpin G-50 columns (GE Healthcare), hybridization was performed in ULTRAhyb Hybridization buffer (Ambion) rotating at 68°C for 4 hr. The membrane was washed twice with 2xSSC buffer supplemented with 0.1%SDS and twice with 0.2xSSC buffer supplemented with 0.1%SDS. The signal was detected by FujiFilm FLA-3000 PhosphorImager and analyzed using ImageJ.