Rnase h point mutant

BM and splenic NK cells were FACS-sorted as NK1.1⁺NKp46⁺CD3⁻CD19⁻ cells, DP and CD11b SP splenic NK cells were sorted as NK1.1⁺NKp46⁺CD11b⁺CD27⁺CD3⁻CD19⁻ and NK1.1⁺NKp46⁺CD11b⁺CD27⁻CD3⁻CD19⁻ cells, respectively. Sorts were performed using FACSAria, BD Biosciences.
Total RNA of the cells was then extracted using the TRIzol® reagent (Ambion, Life Technologies) according to manufacturer’s instructions. Cellular RNA was extracted using RNeasy kits (Qiagen). Contaminating genomic DNA was removed using RNase-free DNase (Qiagen), and cDNA was synthesized using M-MLV RT, RNase H(–) point mutant (Promega, USA) and Random Primers (Invitrogen). Negative control reactions were performed as above, with the omission of the enzyme or the cDNA. cDNA was quantified using the LightCycler 480 SYBR Green I Master (Roche Diagnostics, Germany) on a LightCycler 480 machine (Roche Diagnostics, Switzerland). Standard cycling was used (45 cycles of 95, 60 and 72 °C-steps of 10 s each). Negative control reactions were cycled alongside test samples to ensure the absence of contaminating genomic DNA. Expression was determined relative to the abundance of the housekeeping gene RNA polymerase II Subunit A (Polr2a). Data were analyzed and transcript abundance (Gene/Polr2a) and SD calculated using the LightCycler 480 software release 1.5.0 SP3. The primers used can be found in Supplementary Table 2.

Total RNA was extracted using the TriFast reagent according to the manufacturer's instructions (PEQLAB Biotechnologie GmbH). Annealing with random primers (Life technologies) was performed at 70 °C for 5 min, followed by retrotranscription to complementary (cDNA) with M-MLV RT, RNase H(–) point mutant (Promega) and nucleotides (Roche Diagnostics) by incubating at 40 °C for 10 min, 45 °C for 50 min and 70 °C for 15 min. cDNA was purified with the Wizard SV gel and PCR clean-up system following the manufacturer's instructions (Promega).
cDNA was quantified using the LightCycler 480 SYBR Green I Master (Roche Diagnostics) on a LightCycler 480 machine (Roche Diagnostics). Standard cycling was used (45 cycles of 95, 60 and 72 °C of 10 s each). Expression was determined relative to the housekeeping genes as indicated. Data were analysed, and transcript abundance (gene/housekeeping gene) and s.d. were calculated using the LightCycler 480 software.

Total RNA was extracted using TRIzol (Thermo Fisher Scientific, #15596018) according to the manufacturer’s instructions or by using the MaxWell system (Promega, #AS1340). Isolated RNA was quantified by spectrophotometry and 500 ng of RNA was retrotranscribed to total cDNA using random hexamer and oligo-dT primer mix (Promega, Madison, WI, #C110A), dNTP mix (Promega, #U1511), M-MLV-RT 5X buffer (Promega, #M3681), M-MLV RT, RNase (H–), point mutant (Promega, #M368C) and RNasin-Plus ribonuclease inhibitor (Promega, #N2511). Resulting cDNA was analyzed by TaqMan-based qRT-PCR containing FAM-labeled probes. All reactions were performed in triplicates using a “CFX384 Touch Real-Time PCR Detection System” (Bio-Rad, Hercules, CA). Relative expression levels of candidate genes were acquired by normalization against the housekeeper genes GUSB, HPRT1 and PPIA.