GSM3030317: ET3MEP; Homo sapiens; RNA-Seq
BMMNCs were stained with a lineage-marker cocktail (biotinylated anti-CD2, -CD3, -CD11b, -CD14, -CD15, -CD16, -CD19, -CD56, -CD123, and -CD235a) and subsequently magnetically labeled with Anti-Biotin MicroBeads (Miltenyi Biotec). Lineage-negative stem and progenitor cells (Lin- cells) were obtained by depleting the magnetically-labeled cells after passing through LS columns (Miltenyi Biotec). Lin- cells were stained with antibodies of PE-cy7-conjugated anti-CD38 (BD Biosciences), APC-conjugated anti-CD34 (BD Biosciences), Percp-cy5-conjugated anti-CD45RA (BD Biosciences) and APC-cy7-conjugated anti-CD123 (BD Biosciences). HSCs (CD34+CD38-CD45RA-CD123-) and MEPs (CD34+CD38+CD45RA-CD123-) were sorted with an Aria III instrument (BD Biosciences) Briefly, cells were lysed in lysis buffer (Novogene), and the direct first-strand cDNA was synthesized from polyA-selected RNA with a SMARTer® Ultra Low RNA Kit for Illumina® Sequencing (Clontech) (Cat. #. 634888). cDNAs were purified using AMPure XP beads, followed by LD-PCR, to yield the full-length first-strand cDNA. Double-stranded cDNA was synthesized and amplified with an Advantage® 2 PCR kit. After generating the sequencing libraries using the NEBNext® UltraTM DNA Library Prep Kit for Illumina® (NEB, USA)