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Sample GSM3900158 Query DataSets for GSM3900158
Status Public on Feb 01, 2021
Title POLYA_shWDR82_LPS_45min Replicate2
Sample type SRA
Source name polyA-RNAseq shWdr82_1, LPS 45 min, BMDMs, rep.2
Organism Mus musculus
Characteristics cell type: Primary bone marrow derived macrophages
genotype: wild type
strain: FVB/Hsd
treatment: shWdr82_1, LPS 45 min
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 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 two times pr 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 the Zymo kit with on-column DNAse digestion.
After oligodT-based selection of polyadenylated transcripts from 3-5 ug total RNA, library preparation was performed using the TruSeq Stranded Total RNA sample preparation kit (Illumina RS-122-9007).
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina NextSeq 500
Description polyA-RNAseq shWdr82_1, LPS 45 min, BMDMs, rep.2
Data processing Strand specific paired end reads (76nt) were trimmed for removing the adapter sequence and discarding low quality bases using Trimmomatic v0.27 with PE option (PMID:24695404)
Read quality was then checked for each sample using FastQC v0.11.2 ( High-quality reads were then aligned with TopHat2 v2.1.0 (–very-sensitive --library-type fr-firststrand -r 200 --segment-length 35 --microexon-search --no-mixed --no-discordant -g 1 --coverage-search) (PMID:23618408). Indels due to sequencing errors were identifies using Bowtie2 v.2.6 (PMID: 19261174).
*.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)
Supplementary_files_format_and_content: Strand specific tracks (bigWig)
Submission date Jun 20, 2019
Last update date Feb 02, 2021
Contact name Viviana Piccolo
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 GPL19057
Series (2)
GSE133106 A first exon termination checkpoint that preferentially suppresses extragenic transcription [RNASEQ_BMDM_polyA]
GSE133109 A first exon termination checkpoint that preferentially suppresses extragenic transcription
BioSample SAMN12100456
SRA SRX6099339

Supplementary file Size Download File type/resource 98.8 Mb (ftp)(http) BW 97.0 Mb (ftp)(http) BW
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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