title: GSM5194761: Replicate 2 WT; Mus musculus; RNA-Seq
library_construction_protocol: mRNA-isolation and cDNA transcription were performed immediately after fibroblast sorting from tumors using SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing (Takara) according to the manufacturer's instructions. cDNA was amplified by 16 PCR cycles and samples were purified after cDNA synthesis with AMPure XP beads for PCR Purification (Beckman Coulter). For the determination of cDNA content, a Qubit® 3.0 Fluorometer in combination with Qubit™ 1x dsDNA HS Assay Kit (both Thermo Fisher Scientific) were used. Fragment sizes were determined using High Sensitivity DNA Kit with 2100 Bioanalyzer (both Agilent). The Covaris M220 system was used for controlled cDNA shearing (2 min at 20°C, Peak Power 75W, 20% Duty Factor, Burst Cycle set at 200). Library preparation was performed with SMARTer ThruPLEX DNA-Seq Kit (Takara). Samples were purified after library synthesis with AMPure XP beads for PCR Purification (Beckman Coulter). Six samples were subjected to an indexed single-read sequencing run with 1 x 75 cycles on an Illumina NextSeq™ 500 system.