RNA was isolated from muscle homogenates and reverse transcribed to produce cDNA24 (link). QPCR experiments were designed using established guidelines for experimental design, data normalization and data analysis to maximize the rigor of quantifying the relative levels of mRNA13 (link),66 (link),67 (link). The expression for each gene in control samples was set to 1 and the other expression values were then scaled to that value. PCR primers are listed in Supplementary Table 1 .
Cultured cells were washed twice with ice-cold DPBS and collected in Trizol (Invitrogen). RNA was extracted and isolated with chloroform extraction and isopropyl alcohol precipitation, followed by clean-up with an RNA Clean and Concentrator Kit (Zymo Research). Total RNA was quantified, reverse transcribed, and used for QPCR13 (link).
RNA was isolated from FACS sorted cells by first sorting cells directly into Buffer RLT RNA lysis buffer (Qiagen). RNA was isolated using a Qiagen RNeasy Micro Kit according to the manufacturer’s protocol. RNA yield was quantified using a BioDrop μLite. RNA (50 ng/reaction) was reverse transcribed using a qScript XLT cDNA Supermix (QuantaBio). QPCR experiments were performed on a QuantStudio 5 Real-Time PCR System (Thermo Fisher) with PerfeCTa SYBR Green Supermix, Low Rox (QuantaBio)13 (link).
Cultured cells were washed twice with ice-cold DPBS and collected in Trizol (Invitrogen). RNA was extracted and isolated with chloroform extraction and isopropyl alcohol precipitation, followed by clean-up with an RNA Clean and Concentrator Kit (Zymo Research). Total RNA was quantified, reverse transcribed, and used for QPCR13 (link).
RNA was isolated from FACS sorted cells by first sorting cells directly into Buffer RLT RNA lysis buffer (Qiagen). RNA was isolated using a Qiagen RNeasy Micro Kit according to the manufacturer’s protocol. RNA yield was quantified using a BioDrop μLite. RNA (50 ng/reaction) was reverse transcribed using a qScript XLT cDNA Supermix (QuantaBio). QPCR experiments were performed on a QuantStudio 5 Real-Time PCR System (Thermo Fisher) with PerfeCTa SYBR Green Supermix, Low Rox (QuantaBio)13 (link).
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