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Sample GSM4876568 Query DataSets for GSM4876568
Status Public on Feb 01, 2021
Title HeLa_4sU_empty_rep3
Sample type SRA
 
Source name HeLa
Organism Homo sapiens
Characteristics cell type: HeLa, human cervix, epitheloid carcinoma
treatment: empty vector
Treatment protocol 4-thio-uridine (4sU) was added to the medium at a final concentration of 300 mM for 45 min before harvesting the cells.
Growth protocol HeLa cells were cultured in DMEM containing 10% Foetal Bovine Serum, 1% L-Glutamax solution and 1% Penicillin/Streptomycin solution. Cells were transfected with 2.5 mg of the empty vector or with 2.5 mg of the ZC3H4 C-TERMINAL fragment expression vector.
Extracted molecule total RNA
Extraction protocol Total RNA was extracted using Trizol (Thermofisher cat. 15596026); 4sU-labelled RNA was extracted as described (Austenaa et al. 2015, PMID 26593720)
Library preparation was performed using the TruSeq Stranded Total RNA sample preparation kit (Illumina RS-122-9007) starting from ca. 80-200 ng of nascent 4sU-labelled RNA, without ribosomal depletion.
 
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina NovaSeq 6000
 
Data processing After quality filtering according to the Illumina pipeline and after checking reads quality using FastQC v0.11.2 (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/), strand specific paired end reads (51 bp) were aligned using TopHat v2.1.0 1 (PMID:23618408), allowing up to two mismatches and using the option --b2-very- sensitive --library-type fr-firststrand --no-discordant. Indels due to sequencing errors were identified using Bowtie2 v 2.2.6 (PMID: 19261174). Reads originating from the two strands were separated according to the XS:A:+/XS:A:-flag provided by TopHat2 (PMID:23618408).
SICER v2 (PMID: 24743992) was used to detect the extra-genic transcripts regulated in cells depleted of individual factors. The entire genome was partitioned into blocks of non-overlapping 500 bp windows with a gap size <1000 nt. An effective genome fraction of 1, a fragment size of 0 and a False Discovery Rate (FDR) cut-off <0.01 were used.
Tracks (*.bigWig files) were generated using bamCoverage from deepTools v.3.1.3 (PMID:27079975) (-bs 1 --normalizeUsing RPKM --outFileFormat bigwig -filterRNAstrand forward or --filterRNAstrand reverse)
Genome_build: hg38 (GENCODE) https://www.gencodegenes.org/human/release_33.html
Supplementary_files_format_and_content: Strand specific tracks (bigWig)
 
Submission date Nov 02, 2020
Last update date Feb 02, 2021
Contact name Viviana Piccolo
E-mail(s) viviana.piccolo@ieo.it
Organization name European Institute of Oncology (IEO)
Department Department of Experimental Oncology
Lab Gioacchino Natoli Lab
Street address Via Adamello 16
City Milano
State/province Milano
ZIP/Postal code 20139
Country Italy
 
Platform ID GPL24676
Series (2)
GSE133109 A first exon termination checkpoint that preferentially suppresses extragenic transcription
GSE160658 A first exon termination checkpoint that preferentially suppresses extragenic transcription [RNASEQ_ZC3H4_C]
Relations
BioSample SAMN16624779
SRA SRX9418437

Supplementary file Size Download File type/resource
GSM4876568_RNASEQ_4Su_Hela_empty_2.5_n1_R3_SID103513.FORW.bw 153.8 Mb (ftp)(http) BW
GSM4876568_RNASEQ_4Su_Hela_empty_2.5_n1_R3_SID103513.REV.bw 143.9 Mb (ftp)(http) BW
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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