GSM4703629
GSM4703629: P3Res_L3_sc42; Homo sapiens; RNA-Seq
Fresh bone marrow and peripheral blood mononuclear cells were isolated by the Ficoll-PaqueTM PLUS (GE Healthcare) following the manufacturer's instruction. Bone marrow samples were divided into two parts. One part was used for sorting HSPCs which were stained with human antibodies including lineage cocktail (CD3, CD14, CD16, CD19, CD20, CD56) (BV510, Biolegend), CD34 (APC, Clone 581, BD Biosciences), CD38 (PE-Cy™7, clone HIT2, BD Biosciences), CD90 (PerCP-Cy™5.5, clone5E10, BD Biosciences), CD45RA (FITC, clone HI100, BD Biosciences), CD49f (BV605, clone GoH3, BD Bioscience), CD10 (BV786, clone HI10a, BD Biosciences), CD135 (PE, clone 4G8, BD Biosciences). Another part of bone marrow and all peripheral blood were used for sorting T cells which were stained antibodies including CD3 (APC-Cy™7, clone SK7, BD Biosciences), CD4 (APC, clone RPA-T4, BD Biosciences), CD8 (PE-Cy™7, clone RPA-T8, BD Biosciences). All samples were incubated on ice for 30mins in the dark and washed with PBS. After cell suspension, DAPI (1 ug/ml, Sigma-Aldrich) was added to remove dead cells for FACS. Flow cytometry sorting was performed on a BD FACSAria III (BD Bioscience). Fresh isolated cells were transferred directly into cell lysis buffer and the reverse transcription and template swithc steps were performed following the modified Smart-seq2 protocol. Single cells with different barcodes were pooled for one library. The mixed cDNA were purified using DNA Clean and Concentration Kit (ZYMO, cat.No: D4014), and the small fragments and primers were removed by 0.8X AMPure XP beads (Beckman, cat.No: A63882). 30~40 ng purified products were amplified using biotinylated primers anchored with 8 nt sequences for library indexing. 300~450 ng cDNA was fragmented into 300 bp using Covaris S220 (Covaris). Dynabeads MyOne Streptavidin C1 (Invitrogen, cat.No: 65001) was used to enrich the 3' end of fragmented cDNA. The processes of end repair, polyA-tailing, adaptor ligation and library amplification were using KAPA Hyper Prep Kits (Kapa Biosystems, cat.No: KK8504) and NEBNext Multiplex Oligos for Illumina (Index Primers Set 1) (NEB, cat.No: E7335L). Modified Smart-seq2
GEO
GSM4703629
GEO Accession
GSM4703629