Absolute blue qpcr sybr green mix with low rox

Cells were lysed in TRIzol (Invitrogen, #15596018) and total RNA was isolated using the miRNeasy Mini Kit (Qiagen, #217004). Then cDNA was produced using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, #4368814). Quantitative real-time PCR was performed in triplicate using Power SYBR® Green PCR Master Mix (Applied Biosystems, #4368708) or ABsolute Blue QPCR SYBR Green Mix with low ROX (Thermo Scientific, # AB4322B) on the ABI PRISM® 7500 Real Time PCR System (Applied Biosystems) using the following protocol: 12 minutes at 95°C followed by 40 cycles of 20 seconds at 95°C, 30 seconds at 55°C, and 30 seconds at 70°C. The signal was det ected at 70°C. All primers used for RT-qPCR were listed in Table S5.

Cells were lysed in TRIzol (Thermo Fisher Scientific, 15596018) and total RNA was isolated using the RNeasy Plus Mini Kit (QIAGEN, 74134). High Capacity cDNA Reverse Transcription Kit was used to generate cDNA (Applied Biosystems, 4368814). Quantitative real-time PCR was performed in triplicate using ABsolute Blue QPCR SYBR Green Mix with low ROX (Thermo Fisher Scientific, AB4322B) on the ABI PRISM 7500 Real-Time PCR System (Applied Biosystems). Primers used for qRT-PCR are listed in Table S4.
The quality of RNA was assessed using TapeStation 2200, Agilent Technologies, and 1,000ng of total RNA were used for cDNA Library generation with QuantSeq 3′mRNA-Seq Library Prep Kit FWD (Lexogen, Vienna Austria). According to manufacturer’s recommendations, a common set of external RNA controls were used (ERCC RNA Spike-In mix, ThermoFisher Scientific, 4456740). Samples were submitted to New York Genome Center for SE50 sequencing using a HiSeq 2000. Three independent clonal lines per genotype were used for RNA-seq analysis.

Cell pellets were lysed in TRIzol (Thermo Fisher Scientific, 15596018). RNA was extracted from TRIzol lysate using the RNeasy Mini Kit (Qiagen, 74106). cDNA was produced using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, 43814). Quantitative real-time PCR was performed in triplicate using Absolute Blue QPCR SYBR Green Mix with low ROX (Thermo Fisher Scientific, AB4322B) on the ABI PRISM^® 7500 Real Time PCR System (Applied Biosystems) using the following protocol: 15 minutes at 95 °C followed by 40 cycles of 15 seconds at 95 °C, 30 seconds at 58 °C, and 30 seconds at 72 °C. The signal was detected at 72 °C. All primers used for RT-qPCR are listed in Supplementary Table 5.