title: GSM4661301: NFD 2; Mus musculus; RNA-Seq
library_construction_protocol: Tumoral tissue was dissociated to single cell suspension. Briefly, tumors were digested for 1 hour and half in a Collagenase type V solution (1 mg/ml) at 37°C, 5% CO2 on a rocking platform. Cells digestion was then pelleted and incubated in 2.5% Trypsin for five minutes at 37°C, 5%CO2. Trypsin was then inactivated by complete medium with FBS and cells suspension was further passed through a syringe needle until complete dissociation and filtered through a 40 μm cell strainer. CD45+CD11b+F480+Ly6G- cells were sorted by flow cytometry, resuspended in 1ml PBS plus 0.04% BSA and washed two times by centrifugation at 450 rcf for 7min. After the second wash cells were resuspended in 250 ul of Trizol and RNA was extracted according to standard procedures Total-RNA-sequencing library preparation was performed starting from 0.5 ng of total-RNA with the SMART-Seq Stranded Kit (Clontech-Takara). Libraries were obtained and qualitatively assessed by using TapeStation 4200 and quantified by Qubit Fluorimeter. Afterwards, they were multiplexed in an equimolar pool and sequenced on a NextSeq-500 Illumina Platform generating at least 40 million 75bp-PE reads per sample (i.e. 80 million reads per sample).
attributes.tag: GEO Accession
attributes.value: GSM4661301