Identification of a B-cell restricted progenitor emerging early in fetal development
Identification of a B-cell restricted progenitor emerging early in fetal development
Fetal tissues were dissected under a stereo microscope in Dulbecco's phosphate-buffered saline containing calcium and magnesium (DPBS, Gibco - Thermo Fischer Scientific) supplemented with 10% fetal calf serum (FCS, Sigma-Aldrich, Darmstadt, Germany or Thermo Fisher Scientific). E14.5 fetal livers (FL) were mechanically disrupted by repeated suction using a 27 G needle. Bone marrow cells were prepared from femur, iliac crest and tibial bones collected from individual adult mice. After cleaning of associated tissues, bones were crushed with mortar and pestle, homogenized in DPBS supplemented with 5% FCS and optional 2 mM ethylenediaminetetraacetic acid (EDTA, Sigma-Aldrich), and filtered through a 70 µm cell strainer. Cell aliquots were counted by an automated cell counter (Sysmex KX-21N or Sysmex XP-300 Hematology Analyzer, Sysmex Corporation, Kobe, Japan). All hematopoietic tissues and cell suspensions were always kept on ice during cell preparations. Mini-bulk RNA sequencing was performed using the Smart-Seq2 method as previously described11 with the following modifications. Briefly, 20 cells from each E14.5 FL and adult bone marrow (ABM) progenitor cell population were sorted directly into 4.2 μl lysis buffer containing 2.0 μl 0.4% Triton X-100 (Sigma-Aldrich), 0.1 μl 40 U/μl RNase inhibitor (Takara Bio, San Jose, California, USA), 1 μl 10 mM dNTPs (Thermo Fisher Scientific), 1 μl 10 μM Oligo-dT-ISPCR primer, 0.1 μl 1.07 AU/mL Protease (Qiagen, Hilden, Germany). After 10 min incubation at room temperature, samples were frozen down on dry ice and kept at -80C for up to 3 months. The protease was heat-inactivated by incubation at 72°C for 15 minutes prior to addition of 5.6 μl of reverse transcription master mix containing 2.0 μl 5X Buffer (Clontech - Takara Bio), 2.25 μl RT-PCR Grade Water (Invitrogen - Thermo Fisher Scientific), 0.25 μl RNase Inhibitor (40 U/μl), 0.1 μl TSO-LNA (100 μM, Integrated DNA Technologies, Coralville, Iowa, USA), and 1.0 μl SMARTScribe (100 U/ml, (Takara Bio) per sample. The reverse transcription reaction was performed at 42°C for 90 minutes, followed by 10 cycles of reverse transcription reaction with 50°C for 2 minutes and 42°C for 2 minutes, and a last extension step of 72°C for 15 minutes. Generated cDNA was amplified by the addition of 15 μl amplification mixture containing 12.5 μl 2X SeqAMP Buffer (Takara Bio), 0.125 μl 10 μM ISPCR primer, 0.5 μl 100 U/μl SeqAMP (Takara Bio), and 1.875 μl RT-PCR Grade Water per sample. Amplification was performed using the following PCR conditions: 98°C for 3 minutes, 16 cycles of 98°C for 15 seconds, 67°C for 20 seconds and 72°C for 6 minutes, with a final extension step at 72°C for 5 minutes. Following 1:1 Ampure XP bead purification (Beckman Coulter, Brea, California, United States) and quality check of the cDNA library on an Agilent 2100 Bioanalyzer instrument with High-sensitivity DNA kit (Agilent Technologies, Santa Clara, California, USA), tagmentation reaction was performed with an Illumina Nextera XT DNA sample preparation kit (Illumina, San Diego, California, USA) according to manufacturer's instructions with the following PCR conditions; 72°C for 3 minutes, 12 cycles of 95°C for 30 seconds, 55°C for 30 seconds, and 72°C for 30 seconds, with a final extension step at 72°C for 5 minutes. Tagmented libraries were purified with 0.6:1 ratio Ampure XP beads, quantified by a Qubit fluorometer (Thermo Fisher Scientific) and quality-checked on an Agilent 2100 Bioanalyzer.