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Status |
Public on Feb 01, 2021 |
Title |
shInt11-UT_S13122 |
Sample type |
SRA |
|
|
Source name |
4sU-RNAseq, shInts11_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.
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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.
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Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
Illumina HiSeq 2000 |
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|
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
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|
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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 |
SAMN12100512 |
SRA |
SRX6099344 |