GSM4932851: PreB_WT_3; Mus musculus; RNA-Seq
Bone marrow was flushed in PBS supplemented with 0.1% BSA and 2mM EDTA, filtered through 40μm cell-strainers, and subjected to red blood cell lysis in ACK buffer (0.15M NH4Cl, 10mM KHCO3, 0.1mM EDTA). The samples were stained with: biotin-conjugated antibodies against lineage markers [anti-CD3ε (145-2C11, BioLegend), anti-CD11b (M1/70, BioLegend), anti-CD11c (N418, BioLegend), anti-Ly-6G/Ly-6C (Gr-1) (RB6-8C5, BioLegend), anti-NK-1.1 (PK136, BioLegend), and anti-TER-119 (TER-119, BioLegend)], followed by APC-eFluor 780-conjugated Streptavidin (eBioscience), Alexa Fluor 488-conjugated anti-IgD (11-26c.2a, BioLegend), APC-conjugated anti-CD43 (S7, BD Biosciences), PE-conjugated anti-IgM (II/41, eBioscience), PE-Cy7-conjugated anti-CD19 (6D5, BioLegend), and PerCP-Cy5.5-conjugated anti-CD45R/B220 (RA3-6B2, BioLegend) antibodies. DAPI was added immediately before sorting for dead cell exclusion. Cell sorting was performed on FACSAria and analyzed with FACS Diva software (BD Biosciences). The cells were sorted using FACS into pre-B and immature B cells, gating on B220+CD3-CD11b-NK1.1-CD11c-TER119-Gr1-, and IgM-IgD-CD19+CD43- for pre-B cells, and CD19+IgM+IgD- for immature B cells. RNA was isolated from the FACS-sorted cells using the MagMAX total RNA kit (Ambion, Life Technologies), and quality assessed using Bioanalyzer RNA Pico chips (Agilent). rRNA depletion and library preparation were performed using the SMARTer Stranded RNA-Seq kit (Takara Clontech). The libraries were sequenced on an Illumina HiSeq 4000 sequencer in paired-end 50bp configuration. Cells from multiple mice were pooled to achieve yields of >1.7ng of RNA per sample, and 3-4 independent samples were analyzed per genotype for each cell-type.