title: GSM3539301: Non_injected_CD11b_DCs_III; Mus musculus; RNA-Seq
library_construction_protocol: Mice were injected with LPS/BM-DCs into the footpad and the popliteal and inguinal LNs were collected after 48h. T and B cells were depleted by Dynabeads™ Biotin Binder (Invitrogen™) using Biotin-B220, CD3e, and CD4. The negatively selected cells were sorted for different migratory DC subsets using a BD FACS Aria III (precision: single-cell; nozzle: 100 μm). 100 cells for every population for in total 3 replicates were sorted into individual wells of a 48-well plate (Brand) ﬁlled with 4 µl lysis buffer (Takara). Cells were spun down, immediately chilled to 4°C and stored at −80 °C. All the following experimental steps were performed using the SMART-Seq® v4 Ultra® Low Input RNA Kit (Takara) with a quarter of the recommended reagent volumes. The PCR amplification was performed according to the manual using 21 cycles. Libraries were quantified by QubitTM 3.0 Fluometer (ThermoFisher) and quality was checked using 2100 Bioanalyzer with High Sensitivity DNA kit (Agilent). 0.5 ng of each library was subjected to a tagmentation-based protocol (Nextera XT, Illumina) using a quarter of the recommended reagent volumes, 10 min for tagmentation at 55 °C and 1 min extension time during PCR for multiplexing. After PCR, the libraries were purified using AMPure XP beads and eluted in 15 µl of resuspension buffer. SMART-Seq® v4 Ultra® Low Input RNA Kit (Takara) + Nextera XT (Illumina)
attributes.tag: GEO Accession
attributes.value: GSM3539301