Iscript reverse transcriptase

From total RNA, cDNA was synthesized using iScript Reverse Transcriptase (BioRad, catalog #: 1708841). RT-qPCR assays were performed using SYBR Select Master Mix for CFX (Applied Biosystems) using Thermocycler C1000 CFX384 Real-Time System (BioRad). The primers used for amplification are listed in Supplementary Table 7. Fold changes in transcript levels were calculated as lignan exposure relative to vehicle exposure using the ΔΔC_T method.

Total RNA was isolated from muscle biopsies of 16 subjects, after both the baseline diet and each experimental diet using Trizol extraction followed by clean-up using RNeasy Columns, Qiagen Corp.). The RNA was then DNase treated and quantified using a NanoDrop ND-1000 spectrophotometer. RNA was reverse transcribed into cDNA using iSCRIPT reverse transcriptase (Bio-Rad). Real-time quantitative RT-PCR was performed with 25 ng of cDNA using iQ SYBR Green Supermix (Bio-Rad) on a Bio-Rad Chromo4 Sequence Detection System or an Applied Biosystems CFX1000 (Foster, City, CA). Primer sets (Table 1) were purchased from IDT (Coralville, IA). Levels of gene expression are presented as average expression relative to β-Actin from the same subject as calculated using the ΔΔC_T method [43 (link)-46 (link)]. Briefly, the threshold cycle (C_T) was determined for the gene of interest and β-Actin in each sample and the ΔC_T was calculated for each sample by subtracting the C_T of β-Actin from the C_T of the gene of interest. The ΔΔC_T values were calculated by subtracting the ΔC_T of the experimental samples from the values on the baseline diet. The ΔΔC_T values were then transformed into relative quantity (rq) values using the equation: rq = 2^−ΔΔCT.

PCR Genotyping primers:

Gulo: 5′-CCCAGTGACTAAGGATAAGC-3′, 5′-CGCGCCTTAATTAAGGATCC-3′, 5′-GTCGTGACAGAATGTCTTGC-3′. Wild type band= 343bp, knockout band= 230bp.

Slc23a2⁻: 5′-GGCAGTGTTGGTCCTTCTGT-3′, 5′-CTGGCTATCCTCGTGTCCTG-3′ 5′-CTTAAACCATGGGGCTACCA-3′, 5′-AGACTGCCTTGGGAAAAGCG-3′, wild type band= 140bp, knockout band= 180bp

Tet2^fl and Flt3^ITD genotyping were as previously described ^{27 (link),49 (link)}.
For qPCR, cells were sorted into RLT buffer (Qiagen RNAeasy Micro kit) and RNA was purified according to the manufacturer’s instructions. cDNA was made with iScript reverse transcriptase (BioRad) and quantitative PCR was performed with iTaq Universal SYBR Green (BioRad) and a LightCycler 480 (Roche Applied Science). The signal from each sample was normalized to β-actin. qPCR primers:

Mouse Slc23a2: 5′-GGACAACACCATCCCAGGTA-3′, 5′-CCTTTGCTCACACCCTTCTT-3′.

Mouse Slc23a1: 5′-GAAGCCACCTCAATGAAAGG-3′, 5′-GCTGAGATCTCCAACTCAGGTC-3′.

Mouse β-actin: 5′-CACTGTCGAGTCGCGTCC-3′, 5′-TCATCCATGGCGAACTGGTG-3′

Human SLC23A2: 5′-CTGCAGCCAGCTAGGTCTTG-3′, 5′-AAGCTAGGAGCCCAGGATCA-3′

Human SLC23A1: 5′-TCCTCCTCCTTGGCCTTTGT-3′, 5′-CCCTGGTGGTTTCATGCTGT-3′

Human β-ACTIN: 5′-ATTGGCAATGAGCGGTTC-3′, 5′-CGTGGATGCCACAGGACT-3′

RNA was isolated using a Direct-zol RNA MiniPrep kit (Zymo Research). Total RNA (1 μg) was reverse transcribed using iScript Reverse Transcriptase (BioRad) and qRT-PCR was performed using SSoAdvanced Universal SYBR Green (BioRad). Primer sequences are included in Supplementary Table 1. Samples were run in duplicate using a CFX96 C1000 Touch Real Time Thermal Cycler (Bio-Rad). Data was analyzed using Bio-Rad CFX Manager software.