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Sample GSM5965532

Query DataSets for GSM5965532

Status

Public on Jun 30, 2022

Title

MYCN-overexpressing cell line in long-term culture replicate 2 [MYCN.2826]

Sample type

SRA

Source name

Retina

Organism

Gallus gallus

Characteristics

strain: White Leghorn
genotype: MYCN overexpression
in ovo mycn electroporation: At E3.5
age: E14 dissection

Growth protocol

For MYCN-overexpressing cell lines, the cells were kept in suspension in RPMI 1640 medium supplemented with 10%FBS, 1% MEM-NEAA and 1% Pen/Strep. The medium was changed twice a week.

Extracted molecule

total RNA

Extraction protocol

The central region of E14 chicken retina was dissected and dissociated with 1X Trypsin EDTA. The total RNA was extracted with Qiagen Rneasy Micro Kit (Qiagen, #74004). The RIN value and RNA concentration was measured by TapeStation and 1µg of total RNA was sent for library construction.
MYCN-overexpressing cells were taken and dissociated by vibration and pipetting. The total RNA was extracted with Qiagen Rneasy Micro Kit (Qiagen, #74004). The RIN value and RNA concentration was measured by TapeStation and 1µg of total RNA was sent for library construction.
The library was prepared using the TruSeq stranded mRNA library preparation kit with polyA selection (Illumina Inc.).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

Data processing

Quality check and alignment of the data was done with nf-core/rnaseq pipeline (version 3.4) by adjusting the alignment parameter (--alignEndsProtrude 100 ConcordantPair).
The first 10bp was clipped to avoid biased base composition.
To extract fragment counts, featurecounts (version 2.0.0) (Liao, Smyth, and Shi 2014) was used with minimum mapping quality of 20, and requiring both pairs to be properly aligned on the same chromosome.
Differential expression analysis was done by edgeR (Robinson, McCarthy, and Smyth 2009). Significant differential expressed genes were selected by 1) FDR by the Benjamini and Hochberg method <= 0.05 and 2) |log2 fold-change| >= 1.
For GO/Pathway enrichment analysis, clusterProfiler (version 4.0.6) (Yu et al. 2012) was used. GO gene set enrichment analysis was done by gseGO and at least 10 genes were required to be included in the GO term to be visualized. KEGG gene set enrichment analysis was done by gseKEGG.
Assembly: Galgal6, added transgenes "human MYCN" (Ensembl ID: ENST00000281043.4), "eGFP"
Supplementary files format and content: The file is .txt format and contains normalized gene counts using TMM values with every gene and every sample

Submission date

Mar 22, 2022

Last update date

Jun 30, 2022

Contact name

Finn Hallböök

E-mail(s)

finn.hallbook@igp.uu.se

Organization name

Uppsala University

Department

Dept Immunology, Genetics and Pathology