GSM5051840: P21; Homo sapiens; RNA-Seq
Mononuclear cells from 3 healthy donors and untreated aplastic anemia patients' bone marrow aspirates were isolated using Ficoll density gradient separation and cryopreserved in 90% FBS/ 10% DMSO for storage in liquid nitrogen. The thawing frozen cells were stained with human antibodies including lineage cocktail (CD3, CD14, CD16, CD19, CD20, CD56) (BV510, Biolegend) and CD34 (APC, Clone 581, BD Biosciences) antibodies. Lin-CD34+ HSPCs (cell count ranges from 130 to 2000) were sorted directly into cell lysis buffer on a BD FACSAria III. The reverse transcription and template switch steps were performed following the full-length Smart-seq2 protocol with some modifications. Concentration of oligd(T30VN) and template switch oligo (TSO) primers used for mRNA reverse transcription were double. The cDNA was fragmented using Covaris S220 instead of Tn5 transposase. The processes of end repair, polyA-tailing, adaptor ligation and library amplification were using KAPA Hyper Prep Kits (Kapa Biosystems, Cat# KK8504) and NEBNext Multiplex Oligos for Illumina (Index Primers Set 1) (NEB, Cat# E7335L).The 150bp paired-end reads were generated from Illumina NovaSeq6000 platform. A modified Smart-seq2