|
| Status |
Public on Feb 01, 2021 |
| Title |
POLYA_scr_LPS_45min Replicate1 |
| Sample type |
SRA |
| |
|
| Source name |
polyA-RNAseq shScrambled_1, LPS 45 min, BMDMs_rep.1
|
| Organism |
Mus musculus |
| Characteristics |
cell type: Primary bone marrow derived macrophages
genotype: wild type
strain: FVB/Hsd
treatment: shScrambled_2, 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 shScrambled_1, LPS 45 min, BMDMs_rep.1
|
| 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 (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). 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) https://www.gencodegenes.org/mouse/release_M24.html
Supplementary_files_format_and_content: Strand specific tracks (bigWig)
|
| |
|
| 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 |
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 |
|
| Relations |
| BioSample |
SAMN12100459 |
| SRA |
SRX6099336 |
