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Sample GSM3900128 Query DataSets for GSM3900128
Status Public on Feb 01, 2021
Title sh830-UT_S13381
Sample type SRA
 
Source name 4sU-RNAseq, shWdr82_2, 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 SAMN12100480
SRA SRX6099353

Supplementary file Size Download File type/resource
GSM3900128_mm10_RNASEQ_4Su_sh830_wdr82_UT_hp2_R2.FORW.bw 62.0 Mb (ftp)(http) BW
GSM3900128_mm10_RNASEQ_4Su_sh830_wdr82_UT_hp2_R2.REV.bw 60.2 Mb (ftp)(http) BW
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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