GSM8385656: DOHH2_D40_280920; Homo sapiens; RNA-Seq
Mouse femurs of steady-state (control), PBS-injected (Sham), and DOHH2-injected (grafted) mice were harvested at D19 and D40. BM was flushed and dissociated in PBS containing DNase I (10 U/ml, Pulmozyme, Roche) and Liberase DH (0.025 mg/ml, 5401089001, Roche). Bones were cut into small fragments using scissors in a dissociating media containing 10 U/ml DNase I and Collagenase P (1.2 mg/ml, 11213865001, Roche). Bones and BM were digested at 37°C for 20 min under agitation. PBS supplemented with 5% FBS and 15% sodium citrate was added to neutralize the digestion solution, followed by a centrifugation at 450 g for 5 min. After filtration on 100 µM cell strainer, depletion of red blood cells was carried out with RBC lysis buffer (00-4333-57, Thermo Fisher), stromal cell enrichment was performed using anti-Cd45 (clone 30-F11, 103102, Biolegend) and anti-Ter119 (116202, Biolegend) Abs and using magnetic beads (Dynabeads) according to manufacturer's instructions. After depletion, cells were stained for sorting with the following antibodies: anti-Cd31 BUV395 (clone MEC13.3, BD Biosciences), anti-Cd51 BV650 (clone RMV-7, BD Biosciences), anti-Sca1 PECy7 (clone D7, Biolegend), anti-Cd200 APC (clone OX-90, Biolegend), anti-Cd45 APC-Cy7 (clone 30F11, BD Biosciences), anti-Ter119 APC-Cy7 (116223, Biolegend), anti-hCD45 APC-Cy7 (clone 2D1, BD Biosciences) and anti-hCD20 FITC (clone 2H7, 555622, BD Biosciences) and with live dead reagent (L34959, Thermo). Cells were then sorted using a FACSAria II (BD Biosciences) as Cd45/Ter119negCd31negCd51posCd200pos stromal cells, Cd45/Ter119negCd31posSca1pos endothelial cells31,32, and hCD20pos human lymphoma B cells. After cell sorting, BM DOHH2 were resuspended in RA1 buffer and tris(2-carboxyethyl)phosphine (TCEP) from Macherey-Nagel and stored at -80°C for less than 3 months. RNA was extracted using NucleoSpin RNA kit (Macherey-Nagel) and RNA integrity number (RIN) was assessed using an RNA 6000 Nano kit (5067-1511, Agilent) on a 2100 Bioanalyzer from Agilent. All RIN were above 7. RNA libraries were prepared (500ng of total RNA) for sequencing using standard Illumina Protocols