NextSeq 500 paired end sequencing; RNA-Seq of lymph node fibroblastic reticular cells isolated from bone marrow transplant recipient mice with or without acute Graft-versus-host disease on day 7 or untransplanted control mice
RNA-Seq of lymph node fibroblastic reticular cells isolated from bone marrow transplant recipient mice with or without acute Graft-versus-host disease on day 7 or untransplanted control mice
LN were isolated from recipient mice and digested with Liberase/Dnase at 37°C. Single cell suspension was stained for CD45, gp38 and CD31 to discriminate different lymph node stroma subsets. CD45-CD31-gp38+ FRC were flow sorted and collected in RLT lysis buffer.  RNA was extracted using the RNeasy Micro Kit (QIAGEN, USA) following the manufacturers protocol. RNA yield, quality and integrity were evaluated using the RNA 6000 Pico kit on an Agilent 2100 Bioanalyser (Agilent Technologies, USA). RNA samples were amplified using the SMART Seq v4 Ultra Low Input RNA Kit. Paired-end sequencing libraries were prepared from the amplified cDNA according to the Nextera XT DNA library prep protocol.
Experimental Factor: stimulus
T-cell depleted bone marrow transplant
Experimental Factor: disease
normal