|
| Status |
Public on Nov 07, 2016 |
| Title |
NoDox_12hr_rep1 |
| Sample type |
SRA |
| |
|
| Source name |
hemogenic endothelium (HE)
|
| Organism |
Mus musculus |
| Characteristics |
treatment: none
time point: 12hr
cell type: FLK1+ cells from ESCs differentiated for 48hours
derivative: Ainv18
|
| Treatment protocol |
Cells were treated with and without 1µg/ml doxycyline for 6, 12 and 24 hours before cells were harvested
|
| Growth protocol |
ES cell–derived HE cells were routinely generated by isolating FLK1+ cells from d3.5 embryonic body differentiations. FLK1+ cells where maintained on gelatin coated plates in liquid blast medium (IMDM supplemented with 10% FCS, P/S, 2mM L-Glutamine, 20μg/ml transferrin, 0.45mM MTG, 25μg/ml AA, 15% D4T conditioned medium, 5ng/ml VEGF and 10ng/ml IL6). After 48hous of liquid blast culture cells were treated with and without doxycyline
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated using an Rneasy mini kit according to the manufacturers instructions.
libraries were prepared using 200ng of Total RNA and 14 cycles of amplification in the Agilent Sure Select Automated Strand Specific RNA library prep for Illumina Multiplexed sequencing (G9691-90030) on an Agilent Bravo-BenchCel-MiniHub Automated Liquid Handling Platform. Libraries were sized on an Agilent Bioanalyzer (G2940CA) using a High Sensitivity DNA Kit (5067-4626) and quantified by qPCR using a Kapa Library Quantification Kit for Illumina sequencing platforms (KK4835).
Pooled libraries were sequenced at 1 x 75bp on an Illumina NextSeq500 system (SY-415-1001).
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
NoDox12h_vs_Dox12h
DE genes relative to no dox
|
| Data processing |
bcl2fastq (v2.16.0.10) was used for basecalling and demultiplexing. bcl2fastq parameter: -r 14 -d 14 -p 14 -w 1
Lane-wise alignment was performed by bowtie2 v2.21 to GRCm38 (Ensembl v76) Mus musculus reference genome with default parameters
Generated SAM files from bowtie2 alignment were converted to BAM files by samtools v0.1.19. Parameters for samtools SAM to BAM conversion: -q 10 -F 260.
Resulting lane-wise BAM files from the same sequence library was merged into one BAM. Further, BAM files from the two different batch was merged into one final BAM to be used for downstream analysis.
Read counts from BAM files were extracted under the R environment (R v3.1.0) with the package Rsubread v1.16.1. Count table was loaded to edgeR v3.8.5 and differentially expressed genes between groups were identified by exact test function
Genome_build: GRCm38
Supplementary_files_format_and_content: rawReadCount, RPKM
|
| |
|
| Submission date |
May 16, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Andrew Joshua Lilly |
| E-mail(s) |
zaki.fadlullah@cruk.manchester.ac.uk
|
| Organization name |
CRUK Manchester Institute
|
| Department |
Stem Cell Haematopoiesis
|
| Street address |
Wilmslow Road
|
| City |
Manchester |
| ZIP/Postal code |
M20 4BX |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL19057 |
| Series (1) |
| GSE81466 |
SOX7 supresses the expression of RUNX1 target genes during EHT |
|
| Relations |
| BioSample |
SAMN05001449 |
| SRA |
SRX1763675 |
