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| Status |
Public on Aug 29, 2022 |
| Title |
Spleen_WT_4 |
| Sample type |
SRA |
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| Source name |
spleen
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| Organism |
Mus musculus |
| Characteristics |
tissue: spleen
cell type: B cells
genotype: WT
cohort: 2
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| Extracted molecule |
total RNA |
| Extraction protocol |
Batch1 : Cells were stored in Trizol and extracted using Qiagen mini kit according to recommendations from manufacture; Batch2: Cells were sorted into RLT buffer and extracted using Qiagen mini kit according to recommendations from manufacture
mRNA is purified from approximately 500pg-1ng of total RNA using the Clontech SMARTer Ultra Low V4 RNA Kit. The kit utilizes an oligo(dT) primer, which primes the first-strand synthesis reaction. SPRI beads are used to selectively bind first-strand cDNA. ss cDNA is then amplified by LD PCR to make ds cDNA and is again purified using SPRI beads. During amplification the poly A sequence serves as the universal priming site for end-to-end cDNA amplification. Thus, cDNA without these sequences, such as prematurely terminated cDNAs, containing genomic DNA, or cDNA transcribed from Poly A minus RNA, are not exponentially amplified. The beads are then washed with 80% ethanol and eluted in purification buffer. The full-length cDNAs are then sheared using the Covaris, which results in 200-500bp DNA fragments. The fragmented cDNA library is then end-repaired, A-tailed, and adapters are ligated. Indexed libraries that meet appropriate cut-offs for both are quantified by qRT-PCR using a commercially available kit (KAPA Biosystems) and insert size distribution determined with the LabChip GX. Samples with a yield of ≥0.5 ng/ul are used for sequencing.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
DESeq2_norm_anno_Spleen_only.txt
DESeq2_norm_anno_spleen_vs_vat.txt
190307_7
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| Data processing |
Illumina Casava1.82 software used for basecalling and demultiplexing.
After demultiplexing, raw reads were pseudoaligned to the murine transcriptome (mm10, GENCODE general assembly release vM16) using Kallisto with default settings
Based on the pseudoalignment estimated by Kallisto, transcript levels were quantified and counts were imported into R using tximport. Transcript information was summarized on gene-level. We imported the resulting dataset and performed the downstream analysis using the DESeq2 pipeline.
Genome_build: mm10
Genome_build: transcriptome build: mm10, GENCODE general assembly release vM16
Supplementary_files_format_and_content: Processed DeSeq2 normalized RNA-seq tag counts annotated to the mm10 reference transcriptome
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| Submission date |
Sep 09, 2019 |
| Last update date |
Aug 29, 2022 |
| Contact name |
Joachim Schultze |
| E-mail(s) |
[email protected]
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| Organization name |
LIMES (Life and Medical Sciences Center Genomics and Immunoregulation)
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| Department |
Genomics and Immunoregulation
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| Street address |
Carl-Troll-Strasse 31
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| City |
Bonn |
| State/province |
NRW |
| ZIP/Postal code |
53115 |
| Country |
Germany |
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| Platform ID |
GPL24247 |
| Series (1) |
| GSE137144 |
Splenic and adipose B cells from wild-type and Nlrp3-/- mice |
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| Relations |
| BioSample |
SAMN12718755 |
| SRA |
SRX6821878 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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