title: GSM7679318: Monocyte, tumor, WT4; Mus musculus; RNA-Seq
library_construction_protocol: Tumor tissues were cut into small pieces and digested in RPMI containing collagenase IV (1mg/ml; Worthington Biochemical Corporation), DNase I (40 μg/ml; Roche), and 1% FBS for 30 min at 37 °C. Samples were mixed up and down every 10min. Tubes were centrifuged for a few seconds at 1500rpm and supernatants filtered through a 70µm cell strainer in FACS buffer to stop the reaction. Remaining tissue was further digested in RPMI with collagenase D (1 mg/ml; Roche), DNase I (40 μg/ml; Roche) and 1% of FBS for 20 min at 37 °C, with additional two cycles of repeated pipetting. The solution was filtered through a 70µm cell strainer in in FACS buffer and pool into the first collected tubes.digestion was followed by debris removal using Lympholyte®-M (CEDARLANE) according to manufacturer's instructions. Sorting was based on common markers CD45+Lin (CD19/CD3)-Ly6G-CD11b+ with exclusion of DCs (CD11chi F4/80-), and further separated as macrophages (F4/80+Ly6C-) and monocytes (F4/80-Ly6C+). Total RNA was isolated using miRNeasy Mini Kit (Qiagen). Total RNA was quantified with a Qubit (fluorimeter from Life Technologies) and RNA integrity assessed with a Bioanalyzer (Agilent Technologies). The SMART-Seq v4 kit from Clonetech was used for the reverse transcription and cDNA amplification according to manufacturer's specifications, starting with 1ng (for macrophage) or 10ng (for monocyte) of total RNA as input. 200 pg of cDNA were used for library preparation using the Nextera XT kit from Illumina. Library molarity and quality were assessed with the Qubit and Tapestation using a DNA High sensitivity chip (Agilent Technologies).