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Sample GSM3043279

Query DataSets for GSM3043279

Status

Public on Oct 29, 2018

Title

U2OS cells ZNF768-DN (replicate 3)

Sample type

SRA

Source name

U2OS cells

Organism

Homo sapiens

Characteristics

treatment: ZNF768 inhibition

Treatment protocol

Inhibition of ZNF768 function was achieved by conditional over expression expression of the C-terminal zinc finger of ZNF768 for 12h. For preparation of total RNA cells were resuspended in TRIzol reagent (Life Technologies) at 0.9Mio/ml and snap-frozen.

Extracted molecule

total RNA

Extraction protocol

After thawing RNA was extracted from 0.4ml of TriZol lysate using the direct-zol RNA Miniprep (Zymo Research, Irvine CA, USA) as described in the manufacturer’s protocol. RNA was assessed for purity by UV-vis spectrometry (Nanodrop) and for integrity by Bioanalyzer (Agilent Bioanalyzer 2100, Agilent, Santa Clara USA)). RNA was of high purity (abs. 260/280 >1.9, abs 269/239>2.1) and integrity (Bioanalyzer RIN>9 ) and thus used for further processing.
For production of RNA-seq libraries total RNA was DNAse treated (dsDNAse, Fermentas) and 100 ng of this RNA was processed with a strand-specific protocol (RNA-seq complete kit, NuGEN, San Carlos, USA). In brief the RNA was reverse transcribed to cDNA with a reduced set of hexamer primers, avoiding excessive representation of rRNA in the cDNA. Second strand cDNA synthesis was done in presence of dUTP. After ultrasonic fragmentation of the cDNA and end repair, Illumina-compatible adapter were ligated. Adapters contained uracil in one strand, allowing complete digestion of the second-strand derived DNA. After strand selection the libraries were amplified, assessed for correct insert size on the Agilent Bioanalyser and diluted to 10nM. Barcoded libraries were mixed in equimolar amounts and sequenced on an Illumina HiSeq1500 in single-read mode with a read length of 100 bp.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 1500

Data processing

Reads were mapped against the human genome (GRCh38) and rRNA sequences using ContextMap 2.7.9 with BWA as internal alignment program.
To calculate FPKM (Fragments Per Kilobase Of Exon Per Million Fragments Mapped) values, fragment counts per gene (from Gencode version 25) were calculated using the featureCounts program from the subread package (v1.4.6-p3).
Genome_build: GRCh38
Supplementary_files_format_and_content: read counts for genes

Submission date

Mar 15, 2018

Last update date

Oct 29, 2018

Contact name

Caroline Friedel

Organization name

Ludwig-Maximilians-Universität München

Department

Institut für Informatik

Street address

Amalienstr. 17