title: 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
design: 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
library_construction_protocol: 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.