Superscript 2 enzyme

We isolated RNA from the PBMC of HDs as well as CMV-seropositive donors with the RNeasy Mini (or Micro) kit (Qiagen). For iNKT cells the Arcturus PicoPure RNA Isolation kit (Life Technologies) was used. For the Sézary patients we used the RiboPure blood kit (Life Technologies) to isolate RNA from whole blood. For RNA isolation from murine spleen, the organ was first homogenized using a tissue homogenizer (Qiagen) and then processed with the RNeasy Mini kit (Qiagen). Afterwards, we performed DNA digestion with the Turbo DNA-free kit (Life Technologies). We carried out cDNA synthesis following the manufacturer's instructions using the Superscript II enzyme (Life Technologies) and biotinylated gene-specific primers for the constant gene of the TCR (Supplementary Data 1).
After cDNA synthesis, we carried out RNase H (Life Technologies) treatment and purified the resulting product with the QIAquick PCR purification kit (Qiagen).

Paclitaxel, CITCO, TCPOBOP, androstenol and MTT were obtained from Sigma-Aldrich (St. Louis, MO, USA). Media and reagents for cell culture were acquired from Invitrogen (Carlsbad, CA, USA). Trizol, oligoDT primers, Superscript II enzyme and the Power SYBR Green mastermix were from Life Technologies (Grand Island, NY, USA). Other reagents were of analytical grade. The cell lines used in this experiment were the mouse cell line E9 [21] (link) and the human cell lines A549, H2023, H460, H2030, H1792 and H23 [22] (link). All these cell lines were a gift from Dr. Lucy M. Anderson from the Laboratory of Comparative Carcinogenesis at the Frederick National Laboratory for Cancer Research (United States of America).

Genotyping of the Spot allele was performed using PCR with a standard Taq DNA polymerase (Feldan) and primers flanking the insertion and deletion sites. For semi-quantitative RT-PCR, 10 ng of total RNA was reverse-transcribed using the Superscript II enzyme (Life Technologies, Canada) and a poly-dT₁₇ primer in accordance with manufacturer's instructions. 1 µl of the resulting cDNA pool was then used for amplification of the desired target using specific primers. The expression level of the housekeeping gene Gapdh was used for normalization. PCR consisted of 35 cycles of: 30 s at 95°C, 40 s at 60°C and 30 s at 72°C. Amplicons were resolved on a 2% agarose gel. Primer details can be found in Table S3.

RNA was extracted from human and zebrafish embryos using TRIzol reagents (Life Technologies), following the manufacturer's protocol. For human samples and RT‐qPCR experiments, Superscript II enzyme (Life Technologies) was used for cDNA synthesis. For this set of experiments, a LightCycler 480II (Roche Diagnostics, Basel, Swiss) was used. Probes were selected according to the Software Probe Finder (Roche Diagnostics) and are reported in Table 2. hGUS gene was used as reference gene in human patients and cells derived from healthy donors as standard control. For zebrafish samples, DNase I RNase‐free (Roche Diagnostics) treatment was performed to avoid possible genomic contamination and 1 μg of RNA was reverse‐transcribed using the “ImProm‐II™ Reverse Transcription System” (Promega). RT‐qPCRs were carried out in a total volume of 20 μl containing 1X iQ SYBR Green Supermix (Promega), using proper amount of the RT reaction and a mixture of oligo(dT) and random primers according to manufacturer's instructions. RT‐qPCRs were performed using the Bio‐Rad iCycler iQ Real‐Time Detection System (Bio‐Rad). For normalization purposes, rpl8 expression levels were tested in parallel with the gene of interest. Primers are reported in Table 3. Expression levels in the Y‐axis were relative to the control.

RNA was isolated using RNeasy Mini Kit (Qiagen) from abdominal and GF preadipocytes. Approximately 2 μg was used for library preparation with TruSeq RNA Sample Preparation Kit (Illumina). Following purification, RNA was fragmented with divalent cations at 85 °C, and then cDNA was generated by random primers and SuperScript II enzyme (Life Technologies). Second-strand synthesis was performed followed by end repair, single “A” base addition, and ligation of barcode indexed adaptors to the DNA fragments. Adapter-specific PCRs were performed to generate sequencing libraries. Libraries were size selected with E-Gel EX 2% agarose gels (Life Technologies) and purified by QIAquick Gel Extraction Kit (Qiagen). The result is libraries with inserts ranging in size from 120 to 210 bp with a median size of 155 bp. Libraries were sequenced on HiSeq 2500 instrument.

cDNA libraries for RNA-seq were generated from 100–400 ng total RNA using the TruSeq RNA Sample Preparation Kit (Illumina) or NEBNext Ultra II RNA Library Prep Kit (Illumina), according to the manufacturer’s protocol. Briefly, poly-A tailed RNA molecules were pulled down with poly-T oligo attached magnetic beads. Following purification, mRNA was fragmented with divalent cations at 85°C, and cDNA was generated by random primers and SuperScript II enzyme (Life Technologies). Second strand synthesis was performed, followed by end repair, single "A" base addition, and ligation of barcode-indexed adaptors to the DNA fragments. Adapter specific PCRs were performed to generate sequencing libraries. Libraries were size-selected with E-Gel EX 2% agarose gels (Life Technologies) and purified by the QIAquick Gel Extraction Kit (QIAGEN). Libraries were sequenced on either a HiSeq 2500 or a NextSeq 550 instrument using the NextSeq500/550 High Output Kit v2.5. At least three biological replicates were sequenced for each sorted population.