RNA-seq analysis of hematopoietic stem cells

We isolated bone marrow cells from 8-10-week-old C57BL/6 mice, and stained with antibodies for CD34 (RAM34, eBiosciences, San Diego, CA), Sca-1 (E13-161.7, BD Biosciences Pharmingen, San Jose, CA), c-kit (2B8, BD Biosciences Pharmingen), and lineage marker (Lineage Detection Kit, Miltenyi Biotec Inc., Bergisch Gladbach, Germany). Subsequently, we subjected the stained cells to MoFlo XDP cell sorter system (Beckman Coulter, Fullerton, CA).Following total RNA extractions from 5000 x3 CD34-KSL (c-kit+Sca1+Lin-) LT-HSCs or CD34+KSL ST-HSCs by using ISOGEN (Nippon Gene, Tokyo, Japan), we synthesized cDNA by SMARTer Pico cDNA amplification kit (Clonetech, Mountain View, CA), and amplified them with 20 cycles of PCR. Using the standard protocols from the SOLiD system, we sequenced the amplified cDNA on a SOLiD sequencer (Life Technologies, Carlsbad, CA).