Sybr green brilliant 2 low rox

HPC7 cells were infected with lentivirus containing shRNA specific for STAT5. Scrambled shRNA was used as mock controls. Infected cells were selected using puromycin (1 μg/ml) for 2 days and recovered in IMDM supplemented with 10% FBS and 10% SCF‐conditioned media for 24 h. Total RNA was isolated using RNeasy kit (Qiagen) and subjected to RNA sequencing (BGI). The gene expression level was calculated by using RPKM method (Reads Per kb per Million reads). DEseq packages (Wang et al, 2010) were used for standardization and identification of differentially expressed genes (Anders & Huber, 2010). We defined the differentially expressed genes that satisfy FDR <0.001 and fold change >1.5. Lineage signatures were analyzed by GSEA (Subramanian et al, 2005) using published microarray data of different hematopoietic progenitor cells (Pronk et al, 2007). For RT–PCR, total RNA was reverse‐transcribed into first‐strand cDNA using SuperScriptIII First Strand Synthesis Super Mix (Invitrogen) following the manufacturer's instructions. Quantitative RT–PCR was carried out using SYBR Green Brilliant II low Rox on Stratagene Mx3000P (Agilent Technologies).

Total RNA was isolated using Direct-zol (Zymo Research) and reverse transcribed into cDNA using SuperScript IV First Strand Synthesis System (Invitrogen) following the manufacturer's instructions. Quantitative RT-PCR was carried out using SYBR Green Brilliant II low Rox and a Stratagene Mx3000P machine (Agilent Technologies). For primer sequences, see Supplemental Table S2.