Sm_74-4-4_bio1
Homo sapiens
Protocols: In this study monoclonal cell lines carrying mutations in snoRNA genes were obtained. A number of protospacer sequences were selected for specific cleavage of GAS5 (growth arrest-specific 5) gene. Plasmid pSpCas9(BB)-2A-GFP (pX458) (Addgene, #48138) was used as an expression vector. Human 293FT cell line (Thermo Fisher Scientific) was used in the study. Cells were cultured in DMEM/F12 medium (Thermo Fisher Scientific) containing 10% FBS (Thermo Fisher Scientific) and supplemented with 1x solutions of MEM NEAA (Thermo Fisher Scientific), sodium pyruvate, GlutaMax, and Anti-Anti (Thermo Fisher Scientific) with addition of MycoZap Prophylactic (200 µl per 100 mL of medium) (Lonza) at 37°C and 5% CO2. Small RNA (<200 nucleotide length) was extracted from cells using phenol/chloroform extraction with LRU RNA extraction kit (Biolabmix, Russia) according the manufacturer's protocol and analyzed on a 1.5% TAE-agarose gel and using Bioanalyzer 2100 instrument (Agilent, USA) with an Agilent Small RNA kit. The quality of the Input RNA was assessed by the Agilent Bioanalyzer 2100 System and Agilent Small RNA kit (Agilent, USA). ~0.5 µg of enriched small RNA (<200 nucleotide length) was used for library construction. The construction of cDNA libraries according to a standard protocol using a NEBNext Multiplex Small RNA Library Prep Kit for Illumina (New England Biolabs, UK), as well as massive parallel sequencing on a NextSeq Illumina 500 platform, were conducted at the Institute of Fundamental Medicine and Biology, Kazan Federal University (Kazan, Russia). Libraries concentrations were quantified with the Qubit 2.0 Fluorometric Quantitation system using Qubit DNA HS Assay Kit (Thermo Fisher Scientific). The quality control analysis of libraries was assessed using the Agilent Bioanalyzer 2100 System and DNA High Sensitivity Chip (Agilent, USA).
ENA-FIRST-PUBLIC
2023-11-14T00:40:28Z
ENA-LAST-UPDATE
2023-11-14T00:40:28Z
External Id
SAMEA114568073
INSDC center name
Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, Novosibirsk, Russia
INSDC first public
2023-11-14T00:40:28Z
INSDC last update
2023-11-14T00:40:28Z
INSDC status
public
Submitter Id
E-MTAB-13515:Sm_74-4-4_bio1
broker name
ArrayExpress
cell_line
293FT
cell_type
kidney cell
collection date
not collected
common name
human
developmental stage
embryo stage
disease
-
genotype
CRISPR/Cas9 mediated mutation of GAS5 gene
geographic location (country and/or sea)
not collected
isolate
not applicable
organism part
kidney
replicate
Sm_74-4-4
sample name
E-MTAB-13515:Sm_74-4-4_bio1
scientific_name
Homo sapiens
target gene
GAS5