|
| Status |
Public on May 01, 2016 |
| Title |
cDC2 TG CRE- 2 |
| Sample type |
SRA |
| |
|
| Source name |
Spleen
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57Bl/6
tissue: Spleen
state: Steady State
cell type: cDC2s
genotype/variation: R26-Zeb2Tg/Tg
|
| Treatment protocol |
Steady state mice were used - no treatement
|
| Growth protocol |
Spleens were harvested from mice aged between 6 and 12 weeks
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Spleens were digested in RMPI containing 20mg/ML Liberase and 10U/ml DNase (37oC for 30minutes). Single cell suspensions were filtered through a 70mM strainer and stained with antibodies to allow cDC1s and cDC2s to be isolated by FACS.
RNA concentration and purity were determined spectrophotometrically using the Nanodrop ND-1000 (Nanodrop Technologies) and RNA integrity was assessed using a Bioanalyser 2100 (Agilent). Per sample, an amount of 4 ng of total RNA was used as input for the SMART-Seq v4 Ultra Low Input RNA protocol (version “040215”) from Clontech Laboratories, Inc. Subsequently, 1ng of purified cDNA was sheared to 300bp using the Covaris M220. From the sheared material, sequencing libraries were prepared with the NEBNext Ultra DNA Library Prep Kit for Illumina (version 3.0 -8/15), according to the manufacturer’s protocol including a size selection to 250bp insert size with the following minor modifications: only half of the library is subjected to a 15 cycle PCR in the final library amplification step. Sequence-libraries of each sample were equimolarly pooled and sequenced on 2 NextSeq500 flow-cells at 1x75 bp with a v2 high-output kit.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
cDC2s from R26-Zeb2Tg/Tg number 2
|
| Data processing |
FastQC v0.11.4 is used to check the quality of the samples
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence using Trimmomatic v0.35 with parameters ILLUMINACLIP:2:30:10 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:20 MINLEN:35
Sequenced reads were then mapped to mm10 transcriptome using Tophat2 v2.1.0 with parameters --library-type fr-unstranded --min-intron-length 50 --no-coverage-search
The mapping files were then filtered using samtools v0.1.18 (samtools view with parameters -bq 20) and sorted using samtools sort
Reads were counted using HTSeqCount v0.6.1p1 with parameters -f bam -m intersection-strict -s no -a 10 -t exon -i gene_id
Genome_build: mm10
Supplementary_files_format_and_content: tab-delimited text files including raw counts (output HTSeqCount) for each sample
|
| |
|
| Submission date |
Apr 04, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Liesbet Martens |
| E-mail(s) |
liesbet.martens@irc.vib-ugent.be
|
| Organization name |
VIB-University of Ghent
|
| Department |
VIB Inflammation Research Center
|
| Street address |
Technologiepark 927
|
| City |
Zwijnaarde |
| ZIP/Postal code |
9052 |
| Country |
Belgium |
| |
|
| Platform ID |
GPL19057 |
| Series (1) |
| GSE79903 |
The transcription factor Zeb2 regulates development of conventional and plasmacytoid DCs by repressing Id2 |
|
| Relations |
| BioSample |
SAMN04606224 |
| SRA |
SRX1678309 |
