|
| Status |
Public on Feb 01, 2021 |
| Title |
HeLa_4sU_empty_rep2 |
| 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 |
SAMN16624780 |
| SRA |
SRX9418436 |
