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Sample GSM4109888

Query DataSets for GSM4109888

Status

Public on Jul 01, 2020

Title

RNA-seq_Exp7_KGN_rep2

Sample type

SRA

Source name

KGN cell-line

Organism

Homo sapiens

Characteristics

cell line: KGN
genotype/variation: KGN Cell-line is Heterozygous for FOXL2 C134W mutation

Growth protocol

Cells grown in Ham medium with 10% FCS

Extracted molecule

total RNA

Extraction protocol

Preparation of total mRNA for RNA-seq analysis: Cells were harvested, washed in PBS and pelleted by centrifugation at 300g for 5 minutes. The pellets were kept on ice, resuspended in 350μl RLT Plus-Buffer (Qiagen) with 1% β-mercaptoethanol (Sigma) and passed through a 22G needle 5 times in order to lyse the cells properly. RNA was purified using the RNAeasy Kit (Qiagen) according to manufacturer´s instruction with final elution in 30μl of RNA- free water. The RNA concentration was measured on the NanoDrop 2000 Spectrophotometer (Thermo Scientific). RNA was stored at -80°C.
500ng of total RNA was used for library preparation. Sequencing adaptors were added using the Truseq RNA library prep kit v2 (Illumina) according to the manufacturer’s instructions. Library quality was assessed by analysis on a Bioanalyzer 2100.
RNA and ChIP sequencing libraries were prepared for sequencing using standard Illumina protocols

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NextSeq 500

Description

Exp7_KGN_rep2
Galaxy779-[DESeq2_result_file_on_KGN_TGF_vs_KGN].txt

Data processing

Chip-seq data processing: Fastq datasets were concatenated using the "concatenate” tool (Galaxy tool ID: Cat1, version1.0.0) and trimmed with Trimmomatic version 0.36.0 (with illuminaclip step, Truseq3 (single ended, Mi and Hiseq), max mismatch=2, sliding window, average quality 20). Mapped with Bowtie2 against hg38 using preset settings. Duplicates removed with rmdup.Peak calling with Easeq software: Adaptive Local Thresholding (default settings) with respective knocout samples as negative controls.
RNA-seq data processing: Fastq datasets were concatenated using the "concatenate” tool (Galaxy tool ID: Cat1, version1.0.0) and trimmed with Trimmomatic version 0.36.0 (with illuminaclip step, Truseq3 (single ended, Mi and Hiseq), max mismatch=2, sliding window, average quality 20). Sequencing data was aligned via RNAstar (version2.4.0d-2, single-end reads) using hg38 (Canonical) as refgene and reads were counted using Htseq-count (version 0.6.1, galaxy1 using Union mode and Stranded with a minimum alignment quality of 10). Differential expression was analysed using Deseq2. Genes were called as differentially expressed for expression changes larger than 2-fold and an FDR ≤0.01. A minimum of three biological replicates of each sample were used.
Genome_build: hg38
Supplementary_files_format_and_content: GZIPped WIG file containing ChIP-Seq peaks and output txt files from Deseq2 analysis of RNA seq data. Collumns in Deseq2 data provide the following: Collumn 1: Gene; Collumn 2: Total Counts; Collumn 3: log2 fold difference; Collumn 4: Standard deviation; Collumn 5: Wald Stats; Collumn 6: Wald P value; Collumn 7: Benjamini-Hochberg P value

Submission date

Oct 07, 2019

Last update date

Jul 01, 2020

Contact name

Paul Andreas Compare Cloos

E-mail(s)

[email protected]

Organization name

Copenhagen University

Department

Biotech Research Innovation Centre