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Sample GSM1489183

Query DataSets for GSM1489183

Status

Public on Apr 02, 2015

Title

suz KD R1

Sample type

SRA

Source name

Suz12 knockdown_HSPC

Organism

Mus musculus

Characteristics

strain background: C57BL/6
genotype/variation: Suz12 knockdown
cell type: hematopoietic stem and progenitor cell (HSPC)

Growth protocol

Experimental protocols were approved by the Walter and Eliza Hall Institute Animal Ethics Committee. Retroviral supernatants were prepared as described in Majewski et al., 2008. Gene-specific short hairpin RNA sequences (Supplementary Table) were designed using the DSIR website (http://biodev.cea.fr/DSIR/DSIR.html) (Vert et al., 2006) and subcloned into the LMS-GFP or LMP-BFP vectors that drive expression of a modified microRNA (mir30 backbone) adapted from Dickins et al., 2005 with selectable markers EGFP or EBFP/puromycin, respectively. E14.5 FL cell transduction and transplantation was performed as described in Majewski et al., 2010. Lethally irradiated (11Gy) secondary transplant recipients were injected with 1-3 x 10^6 BM cells isolated from FL primary recipients, with 2 or 3 secondary recipients per donor. Adult BM was harvested from the iliac, femur and tibia bones of CD45.1+ C57BL/6 donor mice. Cells were stained with rat monoclonal antibodies against mature lineage markers (Ter119, B220, CD19, Mac1, Gr1, CD2, CD3, CD8) and then incubated with BioMag goat anti-rat IgG beads (Qiagen) for depletion using a Dynal magnet (Invitrogen). Lineage-depleted cells were stained with fluorophore-conjugated anti-rat IgG, c-Kit and Sca-1 and flow sorted on the FACSAria or BD InFlux (BD Biosciences). Sorted HSPCs were incubated for 16hrs in StemSpan SFEM (StemCell Technologies) with cytokines (50ng/ml SCF, 10ng/ml each of TPO, IL6 and Flt3), placed on viral coated dishes and incubated for a further 24hrs prior to cell collection and intravenous injection into lethally irradiated CD45.2+ recipients with buffer whole BM cells.

Extracted molecule

total RNA

Extraction protocol

Donor+ Lineage- c-Kit+ Sca-1+ GFP+/BFP+ HSPCs were isolated from BM of recipient mice with 30,000 to 100,000 HSPCs pooled from 6 to 10 recipients used per sample. In total, we generated 5 shRNA-Nons, 4 shRNA-Suz12, 4 shRNA- Jarid2.1 and 3 shRNA-Jarid2.2 transduced HSPC samples.
Sequencing libraries were prepared using the TruSeq mRNA Sample Prep Kit (Illumina).
100bp single-end read sequencing was performed on a HiSeq2000 or HiSeq2500 (Illumina) by the Australian Genome Research Facility.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2500

Data processing

The RNA-sequencing reads were aligned to mm10 using TopHat version 2.0.8 with the -- b2-very-sensitive preset
Aligned reads were then summarised over exons using featureCounts from the Rsubread package.
Differential analysis was done using Voom (Law et al., 2014) together with Limma (Smyth, 2004) on genes with a count per million (cpm) larger than 1 in at least 2 samples.
Read counts were divided by total exon length giving RPKM.
Genome_build: mm10
Supplementary_files_format_and_content: csv files with rpkm and differential expression for the groups.

Submission date

Aug 27, 2014

Last update date

May 15, 2019

Contact name

Marnie Blewitt

E-mail(s)

blewitt@wehi.edu.au

Organization name

WEHI

Department

Molecular Medicine Division