PCR Genotyping primers:
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:
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
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′
