NextSeq 550 paired end sequencing; Transcriptome profiling of c-Kit+ hematopoietic stem and progenitor cells enriched from 10-week-old wild-type and Rps19R67∆/R67∆ mutant mice.
Transcriptome profiling of c-Kit+ hematopoietic stem and progenitor cells enriched from 10-week-old wild-type and Rps19R67∆/R67∆ mutant mice.
A single cell suspension of bone marrow from 6 to 8-week-old mice were obtained by flushing tibias and femurs with 1 ml ice-cold Flow Cytometry Buffer (FCB, 1x PBS with 2mM EDTA and 0.5+ACU- biotin-free BSA) using 22-gauge needles. Bone marrow hematopoietic c-kit progenitors were enriched by BD Aria II SORP cell sorter using fluorescence-activated cell sorting with anti-CD117-BV421, anti-Sca1-PE, and anti-lineage (Ter119, Gr1, Mac1, B220, Cd3e) antibody cocktail. Total RNA from FACS-sorted hematopoietic cells was isolated using TRIzol Reagent (Invitrogen) following the manufacturer's RNA isolation protocol. For preparation of NGS libraries polyadenylated mRNA from FASC-sorted c-Kit cells enriched from starting material of 100ng total RNA was processed utilizing NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (New England Biolabs, MA, USA).