title: NextSeq 500 sequencing; Kinome and Rho-GTPase Synthetic Lethal shRNA Library Screen in  paired NF1-/- and GAP-Related Domain Restored Schwann Cells
design: Kinome and Rho-GTPase Synthetic Lethal shRNA Library Screen in  paired NF1-/- and GAP-Related Domain Restored Schwann Cells
library_construction_protocol: Once the cells were confluent, cells were collected using trypsin and split into 3 technical replicates per condition.  At Day 14 post puromycin selection, cells were collected using trypsin and 5 million cells were pelleted and sent to Sigma Aldrich for DNA extraction and sequencing. Patient derived human Schwann cells were grown for 14 days post puromycin selection in 10% FBS in DMEM with 1% L-glutamine and 1% penicillin/streptomycin and passaged once confluent. Cell lines were NF1-/- cells, NF1-/- cells transduced with the GAP related domain of neurofibromin, NF1+/+ cell, and the NF1+/+ cells transduced with an NF1 shRNA. This created two paired cell lines.   An MOI of 0.5 was used with a purchased shRNA library from Sigma Aldrich (CSTVRS-07191316MN) combined with 3-5 clones of each of the human Rho-GTPases.  Puromycin selection was performed to ensure only transduced cells remained for expansion.  Sigma Aldrich performed the nucleic acid extraction. Sigma Aldrich provided the nucleic acid library.