title: GSM4552116: Murine cDC1 cells from MLN - 09; Mus musculus; RNA-Seq
library_construction_protocol: Cells in suspension from murine mesenteric lymph nodes were stained with a cocktail of antibodies to CD45 (30-F11), CD3 (17A2), CD19 (6D5), B220 (RA3-6B2), TER-119 (TER-119), NK-1.1 (PK136), MHC II (IA/I-E) (M5/114.15.2), CD11b (M1/70), CD11c (N418), CD103 (M290), CD64 (X54-5/7.1), F4/80 (BM8), SIRPa (P84), XCR1 (ZET). CD11c+MHCIIhiXCR-1+CD103+ cDC1 were flow cytometry cell sorted on a FACSAriaII (BD Biosciences) after exclusion of dead cells stained by fixable live/dead near-infrared dye (Life technologies) and cell aggregates (identified on FSC-A versus FSC-W scatterplots). Total RNA was isolated using the RNeasy Micro kit (QIAGEN). Briefly, frozen pellets of sorted cells were resuspended in RLT buffer. Buffer and cells were mixed for 30 sec through pipetting and left on the bench for 5 min to assure proper lysis. Extraction was performed according to manufacturer's protocol with an on-column DNAse digestion step added after the first wash buffer step. RNA quality and quantity were measured using the 2100 BioAnalyzer equipped with RNA6000 Pico chip (Agilent Technologies). RNA was subjected to whole transcriptome amplification using Ovation RNA-Seq System V2 (NuGEN) kit, following the manufacturer's protocol. This kit employs a single primer isothermal amplification (SPIA) method to amplify RNA into double stranded cDNA. Amplified cDNA samples were purified using the MinElute Reaction Cleanup kit (Qiagen) according to manufacturer's instructions. Quantity and quality of the cDNA samples were measured using the 2100 BioAnalyzer equipped with DNA1000 chip (Agilent technologies) and the Nanodrop (ThermoFisher Scientific). Libraries to sequence with Illumina platform were constructed the Ovation Ultralow system V2 kit (NuGEN), following manufacturer's instructions. 100ng of amplified cDNA were fragmented by sonication using a Bioruptor Pico (Diagenode). The sheared cDNA was end-repaired to generate blunt ends, then ligated to Illumina adaptors with indexing tags, followed by AMPure XP beads purification. The final size distribution of the libraries was evaluated using 2100 Bioanalyzer equipped with DNA1000 chip (Agilent technologies) and quantified using KAPA library Quantification Kit Illumina platforms (Kapa Biosystems).
attributes.tag: GEO Accession
attributes.value: GSM4552116