|
| Status |
Public on Apr 29, 2019 |
| Title |
DKO_48h_RNA-seq_Rep3 |
| Sample type |
SRA |
| |
|
| Source name |
DKO_48h_RNA-seq
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6J
age: 8-12 weeks
genotype/variation: Tet2/3-DKO (CreERT2, Tet2-flox, Tet3-flox, Rosa26-LSL-YFP)
time point: 48h
tissue: Spleen
|
| Treatment protocol |
Cells were either unstimulated or activated with LPS (25ug/mL) and IL-4 (10ng/mL)
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from naïve and activated B cells with Trizol (ThermoFisher) and the integrity of the RNA was accessed with TapeStation RNA Analysis ScreenTape or Bioanalyzer RNA pico kit (Agilent). 10ng of RNA was reverse transcribed using oligo-dT30 VN primer in the presence of Template Switching Oligo (TSO) with SuperScript II reverse transcriptase. cDNA was pre-amplified with IS PCR primers and PCR products were cleaned up with Ampure XP beads.
One ng of PCR product was used to generate library using NexteraXT library prep kit (Illumina) and tagmentated DNA was amplified with indexed primers for a 12 cycles PCR followed by purification with AmpureXP beads.
RNA-seq, 50bp single end with HiSeq2500
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Description |
Jerry-SMRTSeq-rep2-06_S15
|
| Data processing |
Bcl2fastq v2.20.0.442 was used to obtain fastq files from Illumina
RNA-seq samples at four different time-points collected from WT and DKO conditions were first mapped to the mouse genome mm10/GRCm38 using both hisat2 (PMID: 26355784) (“--no-mixed --no-discordant --add-chrname –dta”) and tophat2 (PMID: 23618408) (“--no-novel-juncs”) alignment programs separately.
Aligned bam files obtained from both the programs were further used to generate the hisat2- and tophat2-specific counts using htseq-count program (PMID: 25260700) (default parameters).
Hisat2- and tophat2-specific count files at each time point for WT and DKO conditions were then used to identify the differentially expressed genes (FDR < 0.05) between matching time points using edgeR program (PMID: 19910308).
The common differentially expressed genes obtained from both hisat2- and tophat2-specific list were used to perform the downstream analysis.
Potential batch effects were removed using svaseq program (PMID: 25294822).
Genome_build: mouse genome mm10 GRCm38 (Dec. 2011) from UCSC
Supplementary_files_format_and_content: tabl delimited files with TPM values for each sample (columns). Column1: chromosome; column2:gene name; column3-22:samples, column23:gene length
|
| |
|
| Submission date |
Jun 25, 2018 |
| Last update date |
Apr 29, 2019 |
| Contact name |
Anjana Rao |
| E-mail(s) |
arao@lji.org
|
| Organization name |
La Jolla Institute for Allergy and Immunology
|
| Department |
Signaling and Gene Expression
|
| Lab |
Anjana Rao
|
| Street address |
9420 Athena Cir
|
| City |
La Jolla |
| State/province |
California |
| ZIP/Postal code |
92037 |
| Country |
USA |
| |
|
| Platform ID |
GPL17021 |
| Series (2) |
| GSE116204 |
TET enzymes augment AID expression via 5hmC modifications at the Aicda superenhancer [RNA-seq] |
| GSE116208 |
TET enzymes augment AID expression via 5hmC modifications at the Aicda superenhancer |
|
| Relations |
| BioSample |
SAMN09479067 |
| SRA |
SRX4290571 |
