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Sample GSM5710637

Query DataSets for GSM5710637

Status

Public on Apr 30, 2023

Title

pre-gRNA_NTC-rep2

Sample type

SRA

Source name

Neuroblastoma SH-SY5Y cells

Organism

Homo sapiens

Characteristics

cell line: SH-SY5Y
cell type: Neuroblastoma cell
treatment: pre-gRNA_none_targeting_control

Treatment protocol

SH-SY5Y cells were transfected at a confluency of 70-80%. For CasRx experiments, the pcDNA3_CasRx-GFP vector (2 mg per 6-well) was co-transfected with either pre-gRNA or gRNA expression plasmids (1 mg per 6-well) using Lipofectamine 3000 kit (5 ml Lipo 3000 and 5 ml P3000 reagents per 6-well; Thermo Fisher Scientific) according to the manufacturer’s guidelines. AgoshRNA expression vectors (2.5 mg per 6-well) were transfected using Lipofectamine 3000 kit (7.5 ml Lipo 3000 and 5 ml P3000 reagents per 6-well; Thermo Fisher Scientific) according to the manufacturer’s instruction. After overnight incubation with the transfection reagents, a media change with fresh DMEM/F12 media (10%FBS, 1%P/S) was performed. RNA samples were harvested 48 hours post-transfection.

Growth protocol

SH-SY5Y cells were maintained in Dulbecco’s modified Eagle’s media (DMEM/F12) (Invitrogen, GIBCO, MA, USA) supplemented with 10% fetal bovine serum (FBS; Invitrogen, GIBCO, MA, USA) and 1% Penicillin-Streptomycin (P/S) (Thermo Fisher Scientific, MA, USA). The cells were passaged before reaching 90% confluency using 0.05% trypsin (Thermo Fisher Scientific). Cells were cultured at 37°C and 5% CO2.

Extracted molecule

polyA RNA

Extraction protocol

RNA extraction was started by lysing the cells in TRIzol reagent (Invitrogen) and followed by the usage of Direct-zol RNA Miniprep Kit (ZYMO RESEARCH, CA, USA) according to the manufacturer’s instructions including DNase on-column treatment for 15 min at room temperature. RNA concentrations and purity were determined using Nanodrop Lite (Thermo Fisher Scientific).
RNA concentrations were measured using the Qubit RNA HS Assay Kit (Thermo Fisher Scientific) on a Qubit 4 Fluorometer (Thermo Fisher Scientific), as well as using Nanodrop Lite (Thermo Fisher Scientific). RNA quality was checked using Agilent Bioanalyzer 2100 (Agilent Technologies) and all RNA integrity (RIN) values exceeded 8.0. Library preparation, and poly(A) RNA sequencing was performed by Beijing Genomics Institute (BGI) in Copenhagen. Briefly, 1 mg total RNA input was used. Non-stranded and polyA-selected RNA library preparation was applied and PE100 sequencing was performed following the specific protocol for sequencing on a DNBSEQ sequencing platform (MGI, Shenzhen, China). Library size and purity control was performed on a Agilent 2100 Bioanalyzer (Agilent Technologies) using a High-Sensitivity DNA-Chip.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

DNBSEQ-T7

Description

Cas13.design1_gtpregRNA_KDpregRNA_NTC.DESeq2.mRNA.txt

Data processing

Filtering and trimming of mRNA-sequencing reads was performed using trim_galore (version 0.6.5dev). The processed reads were mapped to the human reference genome (hg19) using STAR (version 2.7.3a)(Dobin et al., 2013) with default settings. The mapped reads were then quantified per gene annotation using featureCounts (Liao, Smyth and Shi, 2014) with gene annotation from Gencode (version 28). Differential expression analysis (DEA) was based on the featureCounts count data using DESeq2 (Love, Huber and Anders, 2014) in R (http://www.r-project.org).
Genome_build: hg19
Supplementary_files_format_and_content: tab-delimited DESeq2 output text files include raw and normalized counts, gene annotation info, and statistics

Submission date

Dec 01, 2021

Last update date

Apr 30, 2023

Contact name

Karim Rahimi

E-mail(s)

karim@mbg.au.dk

Organization name

Aarhus University

Street address

C.F. Møllers Allé 3