231K-6h
Homo sapiens
Protocols: 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®
ENA-FIRST-PUBLIC
2020-07-01T04:04:17Z
ENA-LAST-UPDATE
2019-07-23T13:42:14Z
External Id
SAMEA5805710
INSDC center name
1) Functional Cancer Genomics Laboratory, Institute of Oncology Research, Bellinzona, Switzerland. 2) Bioinformatics Core Unit, Swiss Institute of Bioinformatics, Bellinzona, Switzerland. 3)Universita della Svizzera italiana, Lugano, Switzerland. 4)Laboratory of Molecular Biology, Istituto di Ricerche Farmacologiche Mario Negri IRCCS, Milano, Italy.
INSDC first public
2020-07-01T04:04:17Z
INSDC last update
2019-07-23T13:42:14Z
INSDC status
public
Submitter Id
E-MTAB-8166:231K-6h
age
4
broker name
ArrayExpress
cell line
SH-SY5Y
cell type
neuroblastoma
common name
human
developmental stage
child
disease
neuroblastoma
genotype
TAU-KO (CRISPR-Cas9) - using gRNA 231 targeting exon2
organism part
cell-line
sample name
E-MTAB-8166:231K-6h
scientific_name
Homo sapiens