cDNA were synthesized by GeneChip T7-Oligo dT) PromoT7-Oligo dTKit (Affymetrix, Inc, Santa Clara, CA) and TaKaRa cDNA Synthesis Kit (TaKaRa Bio Inc, Shiga, JaPromoterm 10 µg total RNA.
WT-hSGLT3 and E457Q-hSGLT3 cDNAs were linearized with XbaI and RNA was transcribed and capped in vitro using the T3 RNA promoter (MEGAscript kit, Ambion).
Double-stranded cDNA was synthesized from 6 µg of total RNA using the SuperScript Choice system (Invitrogen, Carlsbad, CA) and the T7-Oligo (dT) promoter primer kit (Affymetrix, Santa Clara, CA).
The methylation levels at the gene bodies of MGMT, TERT, and TNF were significantly lower in tumors, while both body and promoter of KIT were hypermethylated (Table 2).
Total RNA (≥4 μg) from each group was converted to cDNA by using superscript reverse transcriptase and the T7-Oligo (dT) promoter primer kit (Affymetrix, Inc).
Five independent transposon libraries were constructed in S. Typhimurium ATCC 14028 (STM1) and two in S. Typhi Ty2 (STY1), using the EZ-Tn5 < KAN-2 > Promoter Insertion Kit (Epicentre Biotechnologies) (see methods), and grown in Luria broth (Additional file 1: Table S1).
Subsequently, the quality of RNA was evaluated using an Agilent 2100 Bioanalyzer (Agilent Technologies, Inc., Palo Alto, CA, USA), each RNA sample was converted into double-stranded cDNA by M-MLV ReverseTranscriptase (RNase H free) (TAKARA BIO Inc., Shiga, Japan) using GeneChip T7-Oligo dT) PromoT7-Oligo dTKit.
Methylation array is performed by using the Promoter Methylation Array Kit according to the manufacturer's instructions (Ozyme, France).
First, we used the Fix-Sort-ChIP approach to assess the association of c-Myb with the promoter of the KIT gene in primary human CD34+ hematopoietic progenitors.
We concluded that RHAU regulates spermatogonia differentiation by promoting c-kit expression via directly binding to the G4 DNA motifs c-kit promoter.
