Illumina HiSeq 2000 sequencing; Small RNA-Seq of LSK Slam HSCs MxCre Control vs MxCre Meg3 flox (Meg3 KO)
Small RNA-Seq of LSK Slam HSCs MxCre Control vs MxCre Meg3 flox (Meg3 KO)
Meg3 flox mice were generated by Klibanski et al., loxP sites flank exon 1 to 4 (Klibanski, unpublished). Female Meg3 flox mice were crossed to male MxCre driver mice, generating MxCre Meg3mat-flox/pat-wt mice. Cre+ animals were used in experiments. As control mice, MxCre mice were used. All animals were injected 5 times over 10 days intraperitoneally with pIC (50 µg pIC in PBS) 6-9 weeks after birth. Analysis was started 11 weeks after deletion. Approximately 10000-20000 LSK slam cells (LSK CD150+ CD48-) were FACS sorted into 100 µl QIAzol Lysis Reagent (Qiagen, Venlo, Netherlands).  Total RNA Extraction was performed by adding 20 µl Chloroform (Sigma-Aldrich, St. Louis, MO, USA). Samples were incubated add RT and centrifuged at 12000 rpm for 5 min at RT. The aqueous phase was taken and 0.4 µl glycoblue (Thermo Fisher, Waltham, MA, USA) and 75 µl Isopropanol were added per sample. Samples were stored at -20°C for at least 5 days. Samples were centrifuged at 13000 rpm for 1 h at 4°C. Samples were washed with 70% Ethanol and centrifuged at 13000 rpm for 15 min at 4°C. Supernatant was discarded and the pellet was dried for 1 min. The Pellet was resuspended in 8 µl H2O. RNA quality was analyzed using the Agilent RNA 6000 Pico Kit (Agilent Technologies, Santa Clara, CA, USA) according to manufacturer's instructions. Libraries for small RNA-Seq were generated using the SMARTer® smRNA-Seq Kit for Illumina® (Takara Bio Inc., Kusatsu, Shiga Prefecture, Japan) according to manufacturer's instructions. 23 ng of total RNA was used and 15 cycles were applied for PCR amplification. Quality was checked using the Agilent High Sensitivity DNA Kit (Agilent Technologies, Santa Clara, CA, USA) according to manufacturer's instructions. Size selection was performed by using AMPure XP Beads (Beckman Coulter, Brea, CA, USA) according to manufacturer's instructions.
1 Division of Stem Cells and Cancer, German Cancer Research Center (DKFZ), 69120 Heidelberg, Germany  2 Heidelberg Institute for Stem Cell Technology and Experimental Medicine (HI-STEM gGmbH), 69120 Heidelberg, Germany  3 Faculty of Biosciences, Heidelber
E-MTAB-7519:Cre1_s
Experimental Factor: phenotype
wild type phenotype