GSM4021909: Neo_BM_immB_KO_rep3; Mus musculus; RNA-Seq
BM cells from tet-Lin28b or WT mice fed DOX food for 2 weeks; or 3d old neonatal Lin28b wild-type, heterozygous or knockout mice were extracted by crushing the bones from hind limbs, hips, and spine with mortar and pestle. Adult BM cells were subjected to red blood cell lysis, neonatal BM was not subjected to RBC lysis. Total adult BM cells were then pre-enriched by MACS (Miltenyi Biotec) depletion of Lineage+ cells (Ter119+Gr1+CD11b+CD3e+) according to manufacturer's instructions prior to FACS sorting. Neonatal BM was not pre-enriched before sorting. Antibody staining was performed in FACS buffer (HBSS,supplemented with 0.5% BSA and 2 mM EDTA) at a density of maximum 1x107 cells/100uL volume for 30 min on ice. Immature B cells (CD19+B220loCD93+IgM+CD24hi) were sorted into cold FACS buffer, spun down and resuspended in RNAzol reagent (Sigma-Aldrich). Total RNA was extracted according to the manufacturers instructions. Libraries were generated using the Smartseq V4 Ultra Low input RNA Library Prep Kit (Clontech) and the Nextera XT DNA indexing kit (Illumina) according to the manufacturer's instructions. Libraries were indexed using the Nextera XT v2 indexing kit (Illumina) according to the manufacturers instructions. Libraries were sequenced on an Illumina Nextseq system using a Nextseq V500/550 150cycle high output kit. Reads were aligned using STAR aligner v2.7.1 (80) and counted using RSEM v.1.28.