Illumina HiSeq 4000 sequencing; Cross-species screening platforms identify EPS-8 as a critical link for mitochondrial stress and actin stabilization.
Cross-species screening platforms identify EPS-8 as a critical link for mitochondrial stress and actin stabilization.
NPCs were transduced with lentivirus encoding Cas9 (pHK027-Blast) and selected with Blasticidin. The surviving culture was expanded to 130E6 cells and transduced with lentivirus containing the Avana sgRNA library pools 1-4(62). This library contains 4 sgRNAs for each of 18472 human genes, as well as 663 sgRNAs of random sequence that are not expected to target human genes. The cells were selected using Puromycin for 2 days and were experimentally shown to have a 56% survival rate. The cells were passaged for 2 weeks to allow editing to occur to completion and to allow the protein products time to deplete.  RNA concentration from 3 biological replicates was measured using an Agilent Bioanalyzer at UC Berkeley Functional Genomics Core Lab (FGL). Total RNA (500 ng) was used for library construction using the mRNA HyperPrep Kit (KAPA Biosystems KR1352).
Experimental Factor: organism
Homo sapiens
Experimental Factor: genetic modification
CRISPR Cas9 with Avana sgRNA library pools 1-4 and 663 sgRNAs of random sequence
Experimental Factor: compound
ethanol
Experimental Factor: time
8