title: GSM4661276: Ptenpc-/-;Smad4pc-/- 2; Mus musculus; RNA-Seq
library_construction_protocol: murine prostates were 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 procedure. RNA quality control was performed with the Agilent 2200 Tape Station system and only RNAs having a RIN>8 were used for library preparation. Libraries for mRNA sequencing were prepared starting from 5 ng tot RNA for each sample by using the SMART-Seq v4 Ultra Low Input RNA Kit (Clontech-Takara). Libraries were obtained and qualitatively assessed by using TapeStation 4200 and quantified by Qubit Fluorimeter.  All samples were sequenced on an Illumina NextSeq 500 at an average of 40 million 75-bp single-end reads. 3'mRNA sequecing
attributes.tag: GEO Accession
attributes.value: GSM4661276