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Sample GSM2795209 Query DataSets for GSM2795209
Status Public on Jun 28, 2018
Title Flp8_NoDox_exp3_S202
Sample type SRA
 
Source name Flp-In™ T-Rex Hela cells with doxycycline-inducible EGFP transgene
Organism Homo sapiens
Characteristics cell line: Flp-In T-Rex Hela cells
genotype/variation: doxycycline-inducible EGFP transgene
passage: 8-9
treatment: none
experiment: 3
Treatment protocol Cells were synchronized with a double thymidine arrest (18 hours 2mM thymidine, 9 hours release, 16 hours 2mM thymidine) and after 1 hour release harvested for mRNA isolation. The expression of PP1-NIPP1 wild-type or mutants was induced by the addition of 10 ng/ml Doxycycline to the medium of the cells for the last 13 hours before the cells were harvested.
Growth protocol Cells were grown in low glucose-DMEM medium supplemented with 10% fetal bovine serum and penicilline/streptomycin
Extracted molecule total RNA
Extraction protocol RNA was extracted with the Mammalian Total RNA Miniprep kit (GenElute from SIGMA)
RNA concentration and purity were determined spectrophotometrically using the Nanodrop ND-1000 (Nanodrop Technologies) and RNA integrity was assessed using a Bioanalyzer 2100 (Agilent). Per sample, an amount of 1 microgram of total RNA was used as input. Using the Illumina TruSeq® Stranded mRNA Sample Prep Kit (protocol version: Part # 15031047 Rev. E - October 2013) poly-A containing mRNA molecules were purified from the total RNA input using poly-T oligo-attached magnetic beads. In a reverse transcription reaction using random primers, RNA was converted into first strand cDNA and subsequently converted into double-stranded cDNA in a second strand cDNA synthesis reaction using DNA PolymeraseI and RNAse H. The cDNA fragments were extended with a single ‘A’ base to the 3’ ends of the blunt-ended cDNA fragments after which multiple indexing adapters were ligated introducing different barcodes for each sample. Finally, enrichment PCR was carried out to enrich those DNA fragments that have adapter molecules on both ends and to amplify the amount of DNA in the library. Sequence-libraries of each sample were equimolarly pooled and sequenced on an Illumina NextSeq 500 instrument (High Output, 75 bp, Single Reads, v2) at the VIB Nucleomics core (www.nucleomics.be).
 
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina NextSeq 500
 
Data processing Read Preprocessing: Low quality ends and adapter sequences were trimmed off from the Illumina reads with FastX 0.0.14 and Cutadapt 1.7.1. Using FastX 0.0.14 and ShortRead 1.24.0, we filtered subsequently small reads (length < 35 bp), polyA-reads (more than 90% of the bases equal A), ambiguous reads (containing N) and low quality reads (more than 50% of the bases < Q25). With Bowtie2 v2.2.4 we identified and removed reads that mapped to the spiked-in PhiX. The number of processed reads per sample then varied between 16,086,947 and 20,042,939.
Alignment: Processed reads were aligned with STAR v2.4.1d to the reference genome of Homo sapiens (GRCh38), as downloaded from the Genome Reference Consortium. Default STAR parameter settings were used, except for ‘outSAMprimaryFlag=OneBestScore’, ‘twopassMode=Basic’, ‘alignIntronMin=50’, ‘alignIntronMax=500000’, ‘outSAMtype=BAM’, SortedByCoordinate. Using Samtools 1.1, reads with a mapping quality smaller than 20 were removed from the alignments.
Counting: Transcript coordinates were extracted from the GRC reference annotation with Gffread from the Cufflinks v2.1.1 suite and merged to gene coordinates with mergeBed from the Bedtools v2.17.0 toolkit. GC content and gene length were derived from the gene coordinates. The numbers of aligned reads per gene were summarized using featureCounts 1.4.6 with parameters ‘Q=0’, ‘s=2’, ’t=exon’ and ‘g=gene_id’. We removed 41,248 genes for which all samples had less than 1 count-per-million. As such, we continued with raw counts for 16,985 genes. Raw counts were further corrected within samples for GC-content and between samples using full quantile-normalization, as implemented in the EDASeq package from Bioconductor.
Identifying differential gene expression: With the EdgeR 3.16.5 package, a negative binomial generalized linear model (GLM) was fitted against the normalized counts. We did not use the normalized counts directly, but worked with offsets. Differential expression was tested for with a GLM likelihood ratio test, also implemented in the EdgeR package. The resulting p-values were corrected for multiple testing with Benjamini-Hochberg to control the false discovery rate.
Genome_build: GRCh38
Supplementary_files_format_and_content: One tab-delimited text file with raw read counts per gene and per sample. Gene IDs are Ensembl IDs.
 
Submission date Sep 27, 2017
Last update date May 15, 2019
Contact name Rekin's Janky
E-mail(s) Nucleomics.Bioinformatics@vib.be
Organization name VIB
Department Nucleomics Core
Street address Herestraat 49 Box 816
City Leuven
ZIP/Postal code B-3000
Country Belgium
 
Platform ID GPL18573
Series (1)
GSE104320 PP1-NIPP1 wild type and mutant overexpression in Flip-In™ T-Rex Hela cells
Relations
BioSample SAMN07709954
SRA SRX3220871

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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