|
| Status |
Public on Feb 01, 2021 |
| Title |
shArs2-UT_S13121 |
| Sample type |
SRA |
| |
|
| Source name |
4sU-RNAseq, shArs2_1, untreated BMDMs
|
| Organism |
Mus musculus |
| Characteristics |
cell type: Primary bone marrow derived macrophages
genotype: wild type
strain: FVB/Hsd
|
| Treatment protocol |
Cells were untreated or treated for 45 min with LPS (10 ng/ml) before harvesting. 4-thio-uridine was added to the medium to a final concentration of 150 uM for 45 min before harvesting to allow for incorporation into newly synthesized RNA.
|
| Growth protocol |
Primary bone marrow cells were cultured in DMEM containing 10% North American FBS, 1% L-Glutamax solution, 1% Pen/Strep solution, and 30 % conditioned medium from L929 cells, 0,5% sodium pyruvate and 0,1% b-mercaptoethanol, to allow for differentiation into macrophages for 7 days. Cells under differentiation, from the day after plating, were infected twice per day for two consecutive days with retrovirus produced in Phoenix-ECO cells, containing plasmids expressing either scrambled shRNA or shRNA specific for the mRNA to be depleted. At the end of the second day of infections, puromycin-selection (3,5 ug/ml) were started, and the cells were kept under selection until harvesting at day 7 after plating.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using Trizol whereafter nascent 4sU-labelled RNA was extracted as described in Austenaa et al. 2015 .
Library preparation was performed using the TruSeq Stranded Total RNA sample preparation kit (Illumina RS-122-9007) starting from ca. 100-300 ng of nascent 4sU-labelled RNA, without ribosomal depletion.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| 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 single end reads (51 bp and 76 bp) were aligned to the mm9 reference genomes (Illumina iGenomes reference downloaded from UCSC, http://support.illumina.com/sequencing/sequencing_software/igenome.html) using TopHat v2.1.0 1 (PMID:23618408), allowing up to two mismatches and using the option --b2-very- sensitive --library-type fr-firststrand.
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.
*.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: mm10 (GENCODE) https://www.gencodegenes.org/mouse/release_M24.html
Supplementary_files_format_and_content: .bigWig strand specific tracks
|
| |
|
| Submission date |
Jun 20, 2019 |
| 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 |
GPL13112 |
| Series (2) |
| GSE133105 |
A first exon termination checkpoint that preferentially suppresses extragenic transcription [RNASEQ1_BMDM_4sU] |
| GSE133109 |
A first exon termination checkpoint that preferentially suppresses extragenic transcription |
|
| Relations |
| BioSample |
SAMN12100462 |
| SRA |
SRX6099343 |
