title: JG1
organism: Homo sapiens
description: 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 snoRNA genes encoded within GAS5 (growth arrest-specific 5) introns. 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. Total RNA was isolated from control cells and clones using phenol-chloroform extraction (LIRA reagent from Biolabmix Ltd., Novosibirsk, Russia) according the manufacturer's protocol and analyzed on a 1.5% agarose gel and using an Agilent 2100 Bioanalyzer (Agilent Technologies), with sample with RIN > 8 used. The quality of the Input RNA was assessed by the Agilent Bioanalyzer 2100 System and RNA 6000 Nano Chip (Agilent Technologies). For PolyA mRNA enrichment, high quality RNA with a RIN score > 8 was used. ~1 µg total RNA were used for library construction. PolyA mRNA enrichment and construction of RNA-seq libraries were performed with the NEBNext® Poly(A) mRNA Magnetic Isolation Modulethe and NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina (NEB) correspondingly. Library concentrations were quantified with the Qubit 2.0 Fluorometric Quantitation system using Qubit DNA HS Assay Kit (ThermoFisher Scientific). The quality control analysis of library was assessed using the Agilent Bioanalyzer 2100 System and DNA High Sensitivity Chip (Agilent Technologies).
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2019-10-15T04:05:14Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2019-08-23T11:07:12Z
attributes.tag: External Id
attributes.value: SAMEA5883942
attributes.tag: INSDC center name
attributes.value: Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, Novosibirsk, Russia
attributes.tag: INSDC first public
attributes.value: 2019-10-15T04:05:14Z
attributes.tag: INSDC last update
attributes.value: 2019-08-23T11:07:12Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-8269:JG1
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell line
attributes.value: 293FT
attributes.tag: common name
attributes.value: human
attributes.tag: developmental stage
attributes.value: embryo
attributes.tag: disease
attributes.value: normal
attributes.tag: genotype
attributes.value: empty pX458 plasmid without sgRNA
attributes.tag: organism part
attributes.value: kidney
attributes.tag: sample name
attributes.value: E-MTAB-8269:JG1
attributes.tag: scientific_name
attributes.value: Homo sapiens