GSM1620152: STHSC_WT2; Mus musculus; RNA-Seq
LT-HSCs (LSK CD150+ CD48-), ST-HSCs (LSK CD150- CD48-) and MPPs (LSK CD150- CD48+) from WT and miR-212/132-/- were sorted as follows. Cells were then spun down, resuspended in MACS separating buffer (Phosphate buffered saline, pH7.2, with 0.5% BSA and 2mM EDTA) and filtered through a 70uM cell-strainer. These cells were then blocked with FcBlock (Becton Dickinson) and depleted of mature cells on a magnetic column using biotin-conjugated mouse antibodies for CD3e, CD8, CD4, CD19, B220, CD11b, Gr-1, IL-7Ra, and Ter119 (BioLegend) and streptavidin magnetic beads (Miltenyi), as suggested by the manufacturer (Miltenyi). Cells were subsequently  stained with fluorophore-conjugated antibodies for lineage markers (CD3, CD19, CD11b, Gr-1, Ter119, Nk1.1), cKit, Sca1, CD150, CD48 and with 7-AAD, and several  populations were sorted for analysis including HSCs (Lineage- cKit+ Sca1+ CD150+ CD48-), LSK cells (Lineage- cKit+ Sca1+), and other progenitor subsets. Cells  were sorted on a FACSAria IIu cell sorter (Becton Dickinson) at Caltech. the Caltech Flow Cytometry Core Facility. Mature cell populations were sorted using a  magnetic column using positive-selection for the respective surface marker. Cells were sorted directly into cell lysis buffer and processed using an RNAeasy kit (Qiagen) with DNaseI digestion (Qiagen) as per manufacturer’s protocol.  Libraries were prepared using the SMART-seq2 protocol (Picelli et al., 2013) modified to use Maxima H Minus enzyme for reverse transcription (Thermo Scientific). Amplified cDNA products were purified using AMPure XP SPRI beads (Beckman Coulter) and eluted in TE buffer (Teknova). Cleaned-up amplified cDNA was used for library construction using the Nextera XT DNA Sample Preparation Kit and Nextera XT Index Kit (Illumina). Libraries were then pooled and cleaned-up using AMPure XP SPRI beads (Beckman Coulter Genomics). Final library quality and quantity were assessed using a DNA High Sensitivity chip (Agilent)
GEO Accession
GSM1620152