title: bulkRNA seq of spleens from humanized mice comparing NRG to NSG-DKO strains and additionally vaccination using lentiviral constructs
design: bulkRNA seq of spleens from humanized mice comparing NRG to NSG-DKO strains and additionally vaccination using lentiviral constructs
library_construction_protocol: Immune-reconstitution in the PBL was monitored at 8, 12, 16 and 20 post-HCT. At the endpoint analyses (week 12 or 20), PBL was collected and several tissues were biopsied (spleen, bone marrow, and thymus) and processed as previously reported (Theobald et al, 2020). The tissues were cryopreserved in cryo-medium (40% PBS; 50% Human Serum, Sigma-Aldrich, St. Louis, MO, USA; and 10% DMSO) and stored at -150 °C for further analysis. Spleen cells that were not used for FACS analysis were resuspended in 1 ml Trizol (Invitrogen, Waltham, Massachusetts, USA) and frozen at -80 °C until further use. The RNA was extracted using RNeasy MinElute kit (Qiagen, Hilden, Germany). In detail, the cells frozen in Trizol were thawed on ice and 200 µl chloroform added. The samples were centrifuged at 12,000 g for 12 min at 4 °C. Then aqueous phase was transferred to a new tube and the same volume of 70% ethanol was added. The mixture was transferred to RNeasy MinElute spin column and centrifuged at 8,000 g for 15 second at RT. The flow-through was dumped and 500 µl RPE buffer added. The samples were once again centrifuged at 8,000 g for 15 seconds at room temperature and flow-through was dumped. Then 500 µl 80% ethanol was added and the samples were centrifuge at 8,000 g for 2 min at RT. Afterwards, the samples were dried by centrifugation at max speed for 5 min. The RNA was extracted using 14 µl RNase-free water and centrifugation at max speed for 1 min. The quantity and quality of RNA was checked using photometer (NanoPhotometer N60, Implen, Munich, Germany). Library preparation and NGS analyses were carried out at the production site Cologne (Cologne Center for Genomics (CCG)).