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Sample GSM4872171 Query DataSets for GSM4872171
Status Public on Feb 01, 2021
Title scr-LPS_r3
Sample type SRA
 
Source name Macrophages
Organism Mus musculus
Characteristics cell type: Primary bone marrow derived macrophages
genotype: wild type
strain: FVB/Hsd
treatment: shScrambled_2, LPS 45 min, BMDMs
Treatment protocol Cells were treated for 45 min with LPS (10 ng/ml) before harvesting.
Growth protocol Primary bone marrow cells were cultured in DMEM containing 10% North American origin, low endotoxin Foetal Bovine Serum, 1% L-Glutamax solution, 1% Penicillin/Streptomycin solution, and 30 % conditioned medium from L929 cells and allowed to differentiate for 7 days.
Extracted molecule total RNA
Extraction protocol Total RNA was extracted using Trizol (Thermofisher, cat 15596026) and nascent 4sU-labelled RNA was then extracted as described in 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. 100-300 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 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 Oct 28, 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 GPL24247
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 SAMN16584192
SRA SRX9391079

Supplementary file Size Download File type/resource
GSM4872171_RNASEQ_4Su_BMDM_scr_LPS_expC_R4.FORW_mm10.bw 80.8 Mb (ftp)(http) BW
GSM4872171_RNASEQ_4Su_BMDM_scr_LPS_expC_R4.REV_mm10.bw 77.8 Mb (ftp)(http) BW
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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