Illumina HiSeq 4000 paired end sequencing; RNA-seq of H9 hESC line carrying shRB expression cassette, and H9 hESC line carrying shRB expression cassette in addition to CRISPR/Cas9-mediated nonsense mutations on RBL1 and RBL2
RNA-seq of H9 hESC line carrying shRB expression cassette, and H9 hESC line carrying shRB expression cassette in addition to CRISPR/Cas9-mediated nonsense mutations on RBL1 and RBL2
Cells were grown on 12 well plates for total RNA isolation. Three wells per condition were harvested for technical triplicates. Cell culture medum was aspirated and 250 l of Lysis buffer in GenElute Mammalian Total RNA Miniprep Kit (Sigma-Aldrich) was added directly to the 12 well plates. Cell lysates were transferred to 1.5-ml tubes and were either frozen at -80C or used immediately for RNA extraction. Total RNA was extracted from cells using the GenElute Mammalian Total RNA Miniprep Kit (Sigma-Aldrich) and the On-Column DNase I Digestion set (Sigma-Aldrich).  RNA was poly-A selected and used for library preparation with the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina.