GSM7663538: shKLF2_rep3; Homo sapiens; RNA-Seq
Proliferating regions of IH specimens were minced into pieces and digested with collagenase (Sigma-Aldrich) and Dispase (Gibco). After removal of red blood cells with RBC lysis buffer (Sangon), HemSCs were isolated from cell suspension by anti-CD133 magnetic beads (Miltenyi), and were cultured in vitro on fibronectin-coated (Corning) petri dishes or plates in Endothelial Cell Growth Medium-2 (EGM-2, Lonza, Cat#CC-3162) supplemented with 20% FBS (Gibco). HemSCs between passage 3 to passage 6 were used in this study. Briefly,mRNA was captured from total RNA with VAHTS mRNA Capture Beads (Vazyme, Cat#N401) for fragmentation into short length nucleotides. First-strand cDNA synthesis was then performed with   1st Strand Enzyme Mix 2, followed by generation of second-strand cDNA with 2nd Strand Enzyme Super Mix. Double-stranded cDNA was then ligated with sequencing adapters, purified and amplified with PCR. After size selection, the RNA-seq libraries were sequenced on Illumina Nova Seq 6000 platform at PE150 mode. Three biological replicates of RNA-seq libraries were prepared for shControl and shKLF2 HemSCs respectively.