GSM6337750: Treated: R14M sample; Mus musculus; RNA-Seq
RNA extraction from PB monocytes was performed as previously described (Sanjurjo et al., 2018a). Briefly, 5*105 cells/well cultured in 24-well plates were incubated for 24 h in RPMI medium containing 5% FBS and the indicated stimuli. Cells were then washed with PBS and disrupted with TRIzol reagent (15596026, Thermo Fisher Scientific). Likewise, pellet from cells sorted by flow cytometry (CD45+CD11+Ly6C+Ly6Glow) were also disrupted with TRIzol reagent. For RNA extraction from mouse tumors frozen, tissue was submerged in lysis buffer with β-mercaptoethanol and disrupted mechanically using the homogenizer polytron PRO 200®. In all cases, total RNA was extracted using the RNeasy Mini Kit (74106, Qiagen). Sequencing libraries were prepared following “TruSeq Stranded mRNA Sample Preparation Guide (Part 15031058 Rev. E)” using the “TruSeq® Stranded mRNA Library Prep” kit (20020594, Illumina Inc.) and TruSeq RNA CD Index Plate (96 Indexes, 96 Samples) (20019792, Illumina Inc.). Starting from 150 ng of total RNA, mRNA was purified, fragmented and primed for cDNA synthesis. cDNA first strand was synthesized with SuperScript-II Reverse Transcriptase (18064-014, Thermo Fisher Scientific) for 10 min at 25 °C, 15 min at 42 °C, 15 min at 70 °C and pause at 4 °C. cDNA second strand was synthesized with Illumina reagents at 16 °C for 1 hour. Then, A-tailing and adaptor ligation were performed. Finally, enrichment of libraries was achieved by PCR (30 s at 98 °C; 15 cycles of 10 sec at 98 °C, 30 s at 60 °C, 30 s at 72 °C; 5 min at 72 °C and pause at 4 °C). Afterwards, libraries were visualized on an Agilent 2100 Bioanalyzer using Agilent High Sensitivity DNA kit (5067-4626, Agilent Technologies) and quantified using Qubit dsDNA HS DNA Kit (Q32854, Thermo Fisher Scientific)