Illumina HiSeq 2000 paired end sequencing; Total RNA sequencing for Mus musculus bone marrow lymphoid committed cell populations
Total RNA sequencing for Mus musculus bone marrow lymphoid committed cell populations
C57Bl/6 Ly5.1 Thy1.2 (B6) mice were used at 10-18 week-old (both males and females). They were purchased from CDTA (Orlans, France) and were kept in specific pathogenfree conditions. For cell sorting, antibodies were obtained from BD Pharmingen (San Diego, CA), eBioscience (San Diego, CA), or SONY (Weybridge, UK): anti-NK1.1 (PK136), anti-TCRb (H57-597), anti-CD3 (145-2-C11), anti-Mac 1/CD11b (M1/70), anti-CD19 (1D3), anti-GR 1 (RB6-8C5), anti-Ter 119 (Ly76), anti-Flt 3/CD135 (Flk2; A2F 10.1), anti-c-KIT/CD117 (SCF receptor; 2B8), anti-Sca 1 (E13-161.7), anti-IL7Ra/CD127 (A7R34), anti-VCAM1/CD106 (429), anti-CD138/Sdc1 (MI15), anti-CD93 (AA4.1). They were directly coupled to FITC, APC, APC-eF780, PE, PerCP-Cy5.5, BV421 or conjugated with biotin, the latter being revealed by streptavidin-PE-Cy7. All staining were performed 20 min, at 4C with agitation. After sorting, 1100 cells (CLP CD93+ or CLP CD93-) were seeded on 24-wells plate coated with 6000 OP9 or OP9-DL4 cells per well the day before.  The co-culture was done in Opti-MEM medium (Thermo) supplemented with 10% of FBS (Thermo), 100 U/ml of Penicillin-Streptomycin (Thermo), 50M 2mercaptoethanol, 1ng/ml IL7 (Immunotools), 5ng/ml Flt3-L (Immunotools). BM cells from six mice were collected by crushing femurs, tibia, hips spine bones and pooled.  The cells were incubated with unconjugated rat antibodies against B220, Ter119, Gr1 and Mac1.  Positive cells were magnetically depleted with sheep anti-rat IgG-conjugated beads and sheep anti-mouse IgG-conjugated beads (Dynabeads M-450; Dynal Thermos Fisher Scientific, Villebon, FR). The remaining cells (Lin-, enriched) were labeled with fluorescent conjugated antibodies against c-Kit, Sca-1 and Lin antigens (NK1.1, TCR, CD3, Mac1, CD19, GR1 and Ter119). Lin-Sca-1+c-Kit+ cells are called LSK. Anti-VCAM-1 and anti-Flt3 were additionally used to isolate  different MPP subsets: MPP1 (LSK-Flt3-VCAM-1+), MPP2 (LSK-Flt3+VCAM-1+) and MPP3 (LSK-Flt3+ VCAM-1). The CLP population is characterized by the addition of anti-IL7R.Their phenotype is Lin-Sca-1loc-Kitlo IL7R+ (Figure 1B). Cell sorting was performed on a FACS Aria III upgraded with DIVA software (BD Biosciences, SanDiego, USA). Three independent biological replicates of MPP2, MPP3 and CLP were considered for transcriptome analysis. Pelleted cell were stored at -80C in Qiazol lysis reagent until RNA extraction. Total RNA was extracted using miRNeasy Micro Kit (Qiagen) with DNAse digestion.  NanoDrop spectrophotometer was used to determine total RNA quantity and the 2100 Bioanalyser (Agilent) to qualify total RNA and subsequent libraries. Nuclear and mitochondrial ribosomal RNA was depleted using Ribo-Zero Gold rRNA Removal Kit (Epicentre). Whole transcriptome strand-specific libraries from three progenitors (MPP2, MPP3 and CLP) were performed using SMARTer Stranded RNA-Seq Kit (Clontech) with a total input of [0.11-0.33] g of total RNA per cell population, knowing that rRNA can reach 90% of transcriptome according to cells.