NextSeq 500 paired end sequencing; Tau affects P53 function and cell fate during the DNA damage response
Human neuroblastoma SH-SY5Y cells (94030304, Sigma-Aldrich) were cultured in complete DMEM: Dulbecco's Modified Eagle (61965-059, Gibco) supplemented with 1% non-essential amino acids (11140035, Gibco), 1% penicillin-streptomycin (15140122, Gibco) and 10% fetal bovine serum (10270106, Gibco).Tau-KO clonal lines were generated using the CRISPR/Cas9 DNA-editing technology: The two gRNAs 231 and 232 targeted exon 2 containing the initiating ATG (ENST00000344290.9). Cells in 6-well plates were transfected with the plasmid kindly provided by Dr. Zhang (52961, Addgene) driving expression of one of the two gRNA, Cas9 nuclease and puromycin resistance. One day post-transfection, cells were transferred  to 10 cm plates and incubated for two days with 20 ug/mL puromycin (P8833, Sigma-Aldrich). Single colonies were isolated, amplified, and stored  in liquid nitrogen.  Tau-KO clonal lines were generated using the CRISPR/Cas9 DNA-editing technology: The two gRNAs 231 and 232 targeted exon 2 containing the initiating ATG (ENST00000344290.9). Cells in 6-well plates were transfected with the plasmid kindly provided by Dr. Zhang2 (52961, Addgene) driving expression of one of the two gRNA, Cas9 nuclease and puromycin resistance. One day post-transfection, cells were transferred  to 10 cm plates and incubated for two days with 20 ug/mL puromycin (P8833, Sigma-Aldrich). Single colonies were isolated, amplified, and stored  in liquid nitrogen.   Tau-KO cells 231K and 231P cells were obtained from gRNA 231 while Tau-KO cells 232P were obtained from gRNA 232. The 231A cell line underwent an unsuccessful CRISPR-Cas9 procedure (gRNA 231) and had normal Tau expression Cells were grown at 37°C with saturated humidity and 5% CO2, and maintained in culture for <1 month.  Tau-WT (Parental cells and 231A cells) and Tau-KO (231P, 231K and 232P cells) were treated with 60 uM etoposide for 1, 3, 6, and 16 hours RNA was extracted using the Rnasy kit (Qiagen) and sample quality was assessed using Agilent Bioanalyzer. RNA libraries were prepared for sequencing using NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina®