Kapa sybr fast

Total RNA isolation and cDNA preparation from sorted cells were performed using TRI Reagent™ (12044977, Sigma-Aldrich) and PrimeScript™ RT Reagent Kit (RR037B, Takara Bio, Saint-Germaine-en-Laye, France), respectively, according to the manufacturer’s instruction. cDNA was prepared using 200 ng total RNA and random primers. Quantitative PCR was performed in duplicates with 20 ng template cDNA per reaction using KAPA SYBR^® FAST (KK4603, Roche, Basel, Switzerland) and a CFX96™ Real Time System C1000 Touch™ Thermal Cycler (BioRad, Hercules, CA). Cycling condition was 95°C for 5 min, followed by 40 cycles of 95°C for 15 sec and 60°C for 60 sec. After the completion of PCR reaction, amplification specificity was confirmed by generating dissociation curves. Relative expression values were calculated by ΔΔCT method. First, relative expression levels of Dock2 were calculated as 2^–ΔCt, where ΔCt value represents the difference in Ct values between Dock2 and Gapdh (Ct(Dock2) – Ct(Gapdh)). Then, Dock2 expression levels were normalized to the level in control, DOCK2 non-targeted samples. Following primers were purchased from Microsynth (St. Gallen, Switzerland): Dock2-Fwd, 5’-GGCTCATAGGATTCTCCATCCG-3’; Dock2-Rev, 5’-GATTGGGATGGTGGCTTTCCTG-3’; Gapdh-Fwd, 5’- AGAACATCATCCCTGCATCC-3’; Gapdh-Rev, 5’- TCATCATACTTGGCAGGTTTCTC-3’.

Total RNA from LCLs was extracted using a Direct-Zol RNA MiniPrep kit (Zymo Research, R2052) according to the manufacturer’s instructions. Total RNA from ipMGs was extracted using an miRNeasy micro kit (QIAGEN, 217084) according to the manufacturer’s instructions. Total RNA was reverse transcribed using a High Capacity cDNA Reverse Transcription kit (Applied Biosystems, 4368814), according to manufacturer’s instructions, except for the mRNA stability assay, where an equal volume of RNA (and not equal amounts of RNA) from each sample was used to generate cDNA. qPCR was performed using TaqMan Universal PCR master mix (Applied Biosystems, 4304437) or KAPA SYBR FAST (Roche, KK4605). Primers and TaqMan probes are shown in Supplementary Table 16.

A set of primers in the ABCC1 ORF and in the 3′UTR region (Fig. 1a) were used for qRT-PCR with GAPDH as a loading control. KAPA SYBRFAST (Roche) was used for the qRT-PCR reaction. Normalization was done to GAPDH using the 2^–∆∆Ct method. ABCC1 Primers used for the ORF were Forward Primer 5′-GCTCGTCTTGTCCTGTTTCT-3′ and Reverse Primer 5′-GCATTCCTTCTTCCAGTTCTTTAC-3′. The ABCC1 3′UTR primers were Forward 5′-GCAGTGTTGGTTGCTTACAG-3′ and Reverse 5′-GGATGCCAAGGGAGAGAATTA-3′. The qRT-PCR was done using the CFX96 real time system (BioRad).